Hybrid maize plant and seed 32H20

ABSTRACT

A novel hybrid maize variety designated 32H20 and seed, plants and plant parts thereof, produced by crossing Pioneer Hi-Bred International, Inc. proprietary inbred maize lines. Methods for producing a maize plant that comprises crossing hybrid maize variety 32H20 with another maize plant. Methods for producing a maize plant containing in its genetic material one or more traits introgressed into 32H20 through backcross conversion and/or transformation, and to the maize seed, plant and plant part produced thereby. This invention relates to the hybrid seed 32H20, the hybrid plant produced from the seed, and variants, mutants, and trivial modifications of hybrid 32H20. This invention further relates to methods for producing maize lines derived from hybrid maize variety 32H20 and to the maize lines derived by the use of those methods.

FIELD OF THE INVENTION

This invention relates generally to the field of maize breeding,specifically relating to hybrid maize designated 32H20.

BACKGROUND OF THE INVENTION Plant Breeding

The goal of plant breeding is to combine, in a single variety or hybrid,various desirable traits. For field crops, these traits may includeresistance to diseases and insects, resistance to heat and drought,reducing the time to crop maturity, greater yield, and better agronomicquality. With mechanical harvesting of many crops, uniformity of plantcharacteristics such as germination, stand establishment, growth rate,maturity, and plant and ear height is important. Traditional plantbreeding is an important tool in developing new and improved commercialcrops.

Field crops are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinated if pollen fromone flower is transferred to the same or another flower of the sameplant. A plant is sib pollinated when individuals within the same familyor line are used for pollination. A plant is cross-pollinated if thepollen comes from a flower on a different plant from a different familyor line. The term “cross pollination” and “out-cross” as used herein donot include self pollination or sib pollination.

Plants that have been self-pollinated and selected for type for manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny. A cross between twodifferent homozygous lines produces a uniform population of hybridplants that may be heterozygous for many gene loci. A cross of twoplants each heterozygous at a number of gene loci will produce apopulation of heterogeneous plants that differ genetically and will notbe uniform.

Maize (Zea mays L.), often referred to as corn in the United States, canbe bred by both self-pollination and cross-pollination techniques. Maizehas separate male and female flowers on the same plant, located on thetassel and the ear, respectively. Natural pollination occurs in maizewhen wind blows pollen from the tassels to the silks that protrude fromthe tops of the ears.

The development of a hybrid maize variety in a maize plant breedingprogram involves three steps: (1) the selection of plants from variousgermplasm pools for initial breeding crosses; (2) the selfing of theselected plants from the breeding crosses for several generations toproduce a series of inbred lines, which, individually breed true and arehighly uniform; and (3) crossing a selected inbred line with anunrelated inbred line to produce the hybrid progeny (F1). After asufficient amount of inbreeding successive filial generations willmerely serve to increase seed of the developed inbred. Preferably, aninbred line should comprise homozygous alleles at about 95% or more ofits loci.

During the inbreeding process in maize, the vigor of the linesdecreases. Vigor is restored when two different inbred lines are crossedto produce the hybrid progeny (F1). An important consequence of thehomozygosity and homogeneity of the inbred lines is that the hybridbetween a defined pair of inbreds may be reproduced indefinitely as longas the homogeneity of the inbred parents is maintained. Once the inbredsthat create a superior hybrid have been identified, a continual supplyof the hybrid seed can be produced using these inbred parents and thehybrid corn plants can then be generated from this hybrid seed supply.

32H20 may be used to produce a single cross hybrid, a double crosshybrid, or a three-way hybrid. A single cross hybrid is produced whentwo inbred lines are crossed to produce the F1 progeny. A double crosshybrid is produced from four inbred lines crossed in pairs (A×B and C×D)and then the two F1 hybrids are crossed again (A×B)×(C×D). A three-waycross hybrid is produced from three inbred lines where two of the inbredlines are crossed (A×B) and then the resulting F1 hybrid is crossed withthe third inbred (A×B)×C. In each case, pericarp tissue from the femaleparent will be a part of and protect the hybrid seed.

Large scale commercial maize hybrid production, as it is practicedtoday, requires the use of some form of male sterility system whichcontrols or inactivates male fertility. A reliable method of controllingmale fertility in plants also offers the opportunity for improved plantbreeding. This is especially true for development of maize hybrids,which relies upon some sort of male sterility system. There are severalways in which a maize plant can be manipulated so that is male sterile.These include use of manual or mechanical emasculation (or detasseling),cytoplasmic genetic male sterility, nuclear genetic male sterility,gametocides and the like.

Hybrid maize seed is often produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twoinbred varieties of maize are planted in a field, and the pollen-bearingtassels are removed from one of the inbreds (female) prior to pollenshed. Providing that there is sufficient isolation from sources offoreign maize pollen, the ears of the detasseled inbred will befertilized only from the other inbred (male), and the resulting seed istherefore hybrid and will form hybrid plants.

The laborious detasseling process can be avoided by using cytoplasmicmale-sterile (CMS) inbreds. Plants of a CMS inbred are male sterile as aresult of genetic factors in the cytoplasm, as opposed to the nucleus,and so nuclear linked genes are not transferred during backcrossing.Thus, this characteristic is inherited exclusively through the femaleparent in maize plants, since only the female provides cytoplasm to thefertilized seed. CMS plants are fertilized with pollen from anotherinbred that is not male-sterile. Pollen from the second inbred may ormay not contribute genes that make the hybrid plants male-fertile, andeither option may be preferred depending on the intended use of thehybrid. The same hybrid seed, a portion produced from detasseled fertilemaize and a portion produced using the CMS system can be blended toinsure that adequate pollen loads are available for fertilization whenthe hybrid plants are grown. CMS systems have been successfully usedsince the 1950's, and the male sterility trait is routinely backcrossedinto inbred lines. See Wych, p. 585-586, 1998.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Theseand all patents referred to are incorporated by reference. In additionto these methods, Albertsen et al., of Pioneer Hi-Bred, U.S. Pat. No.5,432,068, describe a system of nuclear male sterility which includes:identifying a gene which is critical to male fertility; silencing thisnative gene which is critical to male fertility; removing the nativepromoter from the essential male fertility gene and replacing it with aninducible promoter; inserting this genetically engineered gene back intothe plant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

These, and the other methods of conferring genetic male sterility in theart, each possess their own benefits and drawbacks. Some other methodsuse a variety of approaches such as delivering into the plant a geneencoding a cytotoxic substance associated with a male tissue specificpromoter or an antisense system in which a gene critical to fertility isidentified and an antisense to that gene is inserted in the plant (seeFabinjanski, et al. EPO 89/3010153.8 Publication No. 329,308 and PCTApplication PCT/CA90/00037 published as WO 90/08828).

Another system useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see Carlson, Glenn R., U.S. Pat. No. 4,936,904). Application ofthe gametocide, timing of the application and genotype specificity oftenlimit the usefulness of the approach and it is not appropriate in allsituations.

The use of male sterile inbreds is but one factor in the production ofmaize hybrids. The development of maize hybrids in a maize plantbreeding program requires, in general, the development of homozygousinbred lines, the crossing of these lines, and the evaluation of thecrosses. Maize plant breeding programs combine the genetic backgroundsfrom two or more inbred lines or various other germplasm sources intobreeding populations from which new inbred lines are developed byselfing and selection of desired phenotypes. Hybrids also can be used asa source of plant breeding material or as source populations from whichto develop or derive new maize lines. Plant breeding techniques known inthe art and used in a maize plant breeding program include, but are notlimited to, recurrent selection, mass selection, bulk selection,backcrossing, making double haploids, pedigree breeding, openpollination breeding, restriction fragment length polymorphism enhancedselection, genetic marker enhanced selection, and transformation. Oftencombinations of these techniques are used. The inbred lines derived fromhybrids can be developed using plant breeding techniques as describedabove. New inbreds are crossed with other inbred lines and the hybridsfrom these crosses are evaluated to determine which of those havecommercial potential. The oldest and most traditional method of analysisis the observation of phenotypic traits but genotypic analysis may alsobe used. Descriptions of breeding methods can also be found in one ofseveral reference books (e.g., Allard, Principles of Plant Breeding,1960; Simmonds, Principles of Crop Improvement, 1979; Fehr, “BreedingMethods for Cultivar Development”, Production and Uses, 2^(nd) ed.,Wilcox editor, 1987).

Backcrossing can be used to improve inbred lines and a hybrid which ismade using those inbreds. Backcrossing can be used to transfer aspecific desirable trait from one line, the donor parent, to an inbredcalled the recurrent parent which has overall good agronomiccharacteristics yet that lacks the desirable trait. This transfer of thedesirable trait into an inbred with overall good agronomiccharacteristics can be accomplished by first crossing a recurrent parentto a donor parent (non-recurrent parent). The progeny of this cross isthen mated back to the recurrent parent followed by selection in theresultant progeny for the desired trait to be transferred from thenon-recurrent parent. Typically after four or more backcross generationswith selection for the desired trait, the progeny will containessentially all genes of the recurrent parent except for the genescontrolling the desired trait. But the number of backcross generationscan be less if molecular markers are used during the selection or elitegermplasm is used as the donor parent. The last backcross generation isthen selfed to give pure breeding progeny for the gene(s) beingtransferred.

Backcrossing can also be used in conjunction with pedigree breeding todevelop new inbred lines. For example, an F1 can be created that isbackcrossed to one of its parent lines to create a BC1. Progeny areselfed and selected so that the newly developed inbred has many of theattributes of the recurrent parent and yet several of the desiredattributes of the non-recurrent parent.

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. The method entails individual plantscross pollinating with each other to form progeny which are then grown.The superior progeny are then selected by any number of methods, whichinclude individual plant, half sib progeny, full sib progeny, selfedprogeny and topcrossing. The selected progeny are cross pollinated witheach other to form progeny for another population. This population isplanted and again superior plants are selected to cross pollinate witheach other. Recurrent selection is a cyclical process and therefore canbe repeated as many times as desired. The objective of recurrentselection is to improve the traits of a population. The improvedpopulation can then be used as a source of breeding material to obtaininbred lines to be used in hybrids or used as parents for a syntheticcultivar. A synthetic cultivar is the resultant progeny formed by theintercrossing of several selected inbreds. Mass selection is a usefultechnique when used in conjunction with molecular marker enhancedselection.

Molecular markers, which includes markers identified through the use oftechniques such as Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), Single NucleotidePolymorphisms (SNPs) and Simple Sequence Repeats (SSRs) may be used inplant breeding methods utilizing 32H20.

One use of molecular markers is Quantitative Trait Loci (QTL) mapping.QTL mapping is the use of markers, which are known to be closely linkedto alleles that have measurable effects on a quantitative trait.Selection in the breeding process is based upon the accumulation ofmarkers linked to the positive effecting alleles and/or the eliminationof the markers linked to the negative effecting alleles from the plant'sgenome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select for the genome of the recurrent parent and against themarkers of the donor parent. Using this procedure can minimize theamount of genome from the donor parent that remains in the selectedplants. It can also be used to reduce the number of crosses back to therecurrent parent needed in a backcrossing program. The use of molecularmarkers in the selection process is often called Genetic Marker EnhancedSelection.

The production of double haploids can also be used for the developmentof inbreds in a breeding program. Double haploids are produced by thedoubling of a set of chromosomes (1N) from a heterozygous plant toproduce a completely homozygous individual. For example, see Wan et al.,“Efficient Production of Doubled Haploid Plants Through ColchicineTreatment of Anther-Derived Maize Callus”, Theoretical and AppliedGenetics, 77:889-892, 1989 and U.S. Patent Application 2003/0005479.This can be advantageous because the process omits the generations ofselfing needed to obtain a homozygous plant from a heterozygous source.

Haploid induction systems have been developed for various plants toproduce haploid tissues, plants and seeds. The haploid induction systemcan produce haploid plants from any genotype by crossing a selected line(as female) with an inducer line. Such inducer lines for maize includeStock 6 (Coe, 1959, Am. Nat. 93:381-382; Sharkar and Coe, 1966, Genetics54:453-464) RWS (see world wide web sitewww.uni-hohenheim.de/%7Eipspwww/350b/indexe.html#Project3), KEMS(Deimling, Roeber, and Geiger, 1997, Vortr. Pflanzenzuchtg 38:203-224),or KMS and ZMS (Chalyk, Bylich & Chebotar, 1994, MNL 68:47; Chalyk &Chebotar, 2000, Plant Breeding 119:363-364), and indeterminategametophyte (ig) mutation (Kermicle 1969 Science 166:1422-1424). Thedisclosures of which are incorporated herein by reference.

Methods for obtaining haploid plants are also disclosed in Kobayashi, M.et al., Journ. of Heredity 71(1):9-14, 1980, Pollacsek, M., Agronomie(Paris) 12(3):247-251, 1992; Cho-Un-Haing et al., Journ. of Plant Biol.,1996, 39(3):185-188; Verdoodt, L., et al., Feb. 1998, 96(2):294-300;Genetic Manipulation in Plant Breeding, Proceedings InternationalSymposium Organized by EUCARPIA, Sept. 8-13, 1985, Berlin, Germany;Chalyk et al., 1994, Maize Genet Coop. Newsletter 68:47; Chalyk, S. T.,1999, Maize Genet. Coop. Newsletter 73:53-54; Coe, R. H., 1959, Am. Nat.93:381-382; Deimling, S. et al., 1997, Vortr. Pflanzenzuchtg 38:203-204;Kato, A., 1999, J. Hered. 90:276-280; Lashermes, P. et al., 1988, Theor.Appl. Genet. 76:570-572 and 76:405-410; Tyrnov, V. S. et al., 1984,Dokl. Akad. Nauk. SSSR 276:735-738; Zabirova, E. R. et al., 1996,Kukuruza I Sorgo N4, 17-19; Aman, M. A., 1978, Indian J. Genet PlantBreed 38:452-457; Chalyk S. T., 1994, Euphytica 79:13-18; Chase, S. S.,1952, Agron. J. 44:263-267; Coe, E. H., 1959, Am. Nat. 93:381-382; Coe,E. H., and Sarkar, K. R., 1964 J. Hered. 55:231-233; Greenblatt, I. M.and Bock, M., 1967, J. Hered. 58:9-13; Kato, A., 1990, Maize Genet.Coop. Newsletter 65:109-110; Kato, A., 1997, Sex. Plant Reprod.10:96-100; Nanda, D. K. and Chase, S. S., 1966, Crop Sci. 6:213-215;Sarkar, K. R. and Coe, E. H., 1966, Genetics 54:453-464; Sarkar, K. R.and Coe, E. H., 1971, Crop Sci. 11:543-544; Sarkar, K. R. and Sachan J.K. S., 1972, Indian J. Agric. Sci. 42:781-786; Kermicle J. L., 1969,Mehta Yeshwant, M. R., Genetics and Molecular Biology, September 2000,23(3):617-622; Tahir, M. S. et al. Pakistan Journal of Scientific andIndustrial Research, August 2000, 43(4):258-261; Knox, R. E. et al.Plant Breeding, August 2000, 119(4):289-298; U.S. Pat. No. 5,639,951 andU.S. patent application Ser. No. 10/121,200, the disclosures of whichare incorporated herein by reference.

Hybrid seed production requires elimination or inactivation of pollenproduced by the female parent. Incomplete removal or inactivation of thepollen provides the potential for self-pollination. This inadvertentlyself-pollinated seed may be unintentionally harvested and packaged withhybrid seed. Also, because the male parent is grown next to the femaleparent in the field there is the very low probability that the maleselfed seed could be unintentionally harvested and packaged with thehybrid seed. Once the seed from the hybrid bag is planted, it ispossible to identify and select these self-pollinated plants. Theseself-pollinated plants will be genetically equivalent to one of theinbred lines used to produce the hybrid. Though the possibility ofinbreds being included in a hybrid seed bag exists, the occurrence isvery low because much care is taken by seed companies to avoid suchinclusions. It is worth noting that hybrid seed is sold to growers forthe production of grain and forage and not for breeding or seedproduction. By an individual skilled in plant breeding, these inbredplants unintentionally included in commercial hybrid seed can beidentified and selected due to their decreased vigor when compared tothe hybrid. Inbreds are identified by their less vigorous appearance forvegetative and/or reproductive characteristics, including shorter plantheight, small ear size, ear and kernel shape, cob color, or othercharacteristics.

Identification of these self-pollinated lines can also be accomplishedthrough molecular marker analyses. See, “The Identification of FemaleSelfs in Hybrid Maize: A Comparison Using Electrophoresis andMorphology”, Smith, J. S. C. and Wych, R. D., Seed Science andTechnology 14, pages 1-8 (1995), the disclosure of which is expresslyincorporated herein by reference. Through these technologies, thehomozygosity of the self pollinated line can be verified by analyzingallelic composition at various loci along the genome. Those methodsallow for rapid identification of the invention disclosed herein. Seealso, “Identification of Atypical Plants in Hybrid Maize Seed byPostcontrol and Electrophoresis” Sarca, V. et al., Probleme de GeneticaTeoritica si Aplicata Vol. 20 (1) pages 29-42.

Another form of commercial hybrid production involves the use of amixture of male sterile hybrid seed and male pollinator seed. Whenplanted, the resulting male sterile hybrid plants are pollinated by thepollinator plants. This method is primarily used to produce grain withenhanced quality grain traits, such as high oil, because desired qualitygrain traits expressed in the pollinator will also be expressed in thegrain produced on the male sterile hybrid plant. In this method thedesired quality grain trait does not have to be incorporated by lengthyprocedures such as recurrent backcross selection into an inbred parentline. One use of this method is described in U.S. Pat. Nos. 5,704,160and 5,706,603.

There are many important factors to be considered in the art of plantbreeding, such as the ability to recognize important morphological andphysiological characteristics, the ability to design evaluationtechniques for genotypic and phenotypic traits of interest, and theability to search out and exploit the genes for the desired traits innew or improved combinations.

The objective of commercial maize hybrid line development resulting froma maize plant breeding program is to develop new inbred lines to producehybrids that combine to produce high grain yields and superior agronomicperformance. One of the primary traits breeders seek is yield. However,many other major agronomic traits are of importance in hybridcombination and have an impact on yield or otherwise provide superiorperformance in hybrid combinations. Such traits include percent grainmoisture at harvest, relative maturity, resistance to stalk breakage,resistance to root lodging, grain quality, and disease and insectresistance. In addition, the lines per se must have acceptableperformance for parental traits such as seed yields, kernel sizes,pollen production, all of which affect ability to provide parental linesin sufficient quantity and quality for hybridization. These traits havebeen shown to be under genetic control and many if not all of the traitsare affected by multiple genes.

A breeder uses various methods to help determine which plants should beselected from the segregating populations and ultimately which inbredlines will be used to develop hybrids for commercialization. In additionto the knowledge of the germplasm and other skills the breeder uses, apart of the selection process is dependent on experimental designcoupled with the use of statistical analysis. Experimental design andstatistical analysis are used to help determine which plants, whichfamily of plants, and finally which inbred lines and hybrid combinationsare significantly better or different for one or more traits ofinterest. Experimental design methods are used to assess error so thatdifferences between two inbred lines or two hybrid lines can be moreaccurately determined. Statistical analysis includes the calculation ofmean values, determination of the statistical significance of thesources of variation, and the calculation of the appropriate variancecomponents. Either a five or one percent significance level iscustomarily used to determine whether a difference that occurs for agiven trait is real or due to the environment or experimental error. Oneof ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr, Walt, Principles of CultivarDevelopment, pages 261-286 (1987) which is incorporated herein byreference. Mean trait values may be used to determine whether traitdifferences are significant, and preferably the traits are measured onplants grown under the same environmental conditions.

SUMMARY OF THE INVENTION

According to the invention, there is provided a hybrid maize plant, andits parts designated as 32H20, produced by crossing two Pioneer Hi-BredInternational, Inc. proprietary inbred maize lines GE024207702 andGE2543998. These lines, deposited with the American Type CultureCollection, (ATCC), Manassas, Va. 20110, have Accession Number ______for GE024207702 and Accession Number ______ for GE2543998. Thisinvention thus relates to the hybrid seed 32H20, the hybrid plant andits parts produced from the seed, and variants, mutants and trivialmodifications of hybrid maize 32H20. This invention also relates toprocesses for making a maize plant containing in its genetic materialone or more traits introgressed into 32H20 through backcross conversionand/or transformation, and to the maize seed, plant and plant partproduced by such introgression. This invention further relates tomethods for producing maize lines derived from hybrid maize 32H20 and tothe maize lines derived by the use of those processes. This hybrid maizeplant is characterized by resistance to Grey Leaf Spot and Goss's Wilt,and good staygreen.

Definitions

Certain definitions used in the specification are provided below. Alsoin the examples that follow, a number of terms are used herein. In orderto provide a clear and consistent understanding of the specification andclaims, including the scope to be given such terms, the followingdefinitions are provided. NOTE: ABS is in absolute terms and % MN ispercent of the mean for the experiments in which the inbred or hybridwas grown. PCT designates that the trait is calculated as a percentage.% NOT designates the percentage of plants that did not exhibit a trait.For example, STKLDG % NOT is the percentage of plants in a plot thatwere not stalk lodged. These designators will follow the descriptors todenote how the values are to be interpreted. Below are the descriptorsused in the data tables included herein.

ABTSTK=ARTIFICIAL BRITTLE STALK. A count of the number of “snapped”plants per plot following machine snapping. A snapped plant has itsstalk completely snapped at a node between the base of the plant and thenode above the ear. Expressed as percent of plants that did not snap.

ADF=PERCENT ACID DETERGENT FIBER. The percent of dry matter that is aciddetergent fiber in chopped whole plant forage.

ALLELE. Any of one or more alternative forms of a genetic sequence.Typically, in a diploid cell or organism, the two alleles of a givensequence typically occupy corresponding loci on a pair of homologouschromosomes.

ALTER. The utilization of up-regulation, down-regulation, or genesilencing.

ANTHESIS. The time of a flower's opening.

ANT ROT=ANTHRACNOSE STALK ROT (Colletotrichum graminicola). A 1 to 9visual rating indicating the resistance to Anthracnose Stalk Rot. Ahigher score indicates a higher resistance. Data are collected only whensufficient selection pressure exists in the experiment measured.

BACKCROSSING. Process in which a breeder crosses a hybrid progeny lineback to one of the parental genotypes one or more times.

BARPLT=BARREN PLANTS. The percent of plants per plot that were notbarren (lack ears).

BORBMN=ARTIFICIAL BRITTLE STALK MEAN. The mean percent of plants not“snapped” in a plot following artificial selection pressure. A snappedplant has its stalk completely snapped at a node between the base of theplant and the node above the ear. Expressed as percent of plants thatdid not snap. A high number is good and indicates tolerance to brittlesnapping.

BRENGMN=BRITTLE STALK ENERGY MEAN. The mean amount of energy per unitarea needed to artificially brittle snap a corn stalk. A high number isgood and indicates tolerance to brittle snapping.

BREEDING. The genetic manipulation of living organisms.

BREEDING CROSS. A cross to introduce new genetic material into a plantfor the development of a new variety. For example, one could cross plantA with plant B, wherein plant B would be genetically different fromplant A. After the breeding cross, the resulting F1 plants could then beselfed or sibbed for one, two, three or more times (F1, F2, F3, etc.)until a new inbred variety is developed. For clarification, such newinbred varieties would be within a pedigree distance of one breedingcross of plants A and B. The process described above would be referredto as one breeding cycle.

BRTSTK=BRITTLE STALKS. This is a measure of the stalk breakage near thetime of pollination, and is an indication of whether a hybrid or inbredwould snap or break near the time of flowering under severe winds. Dataare presented as percentage of plants that did not snap. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

CELL. Cell as used herein includes a plant cell, whether isolated, intissue culture or incorporated in a plant or plant part.

CLDTST=COLD TEST. The percent of plants that germinate under cold testconditions.

CLN=CORN LETHAL NECROSIS. Synergistic interaction of maize chloroticmottle virus (MCMV) in combination with either maize dwarf mosaic virus(MDMV-A or MDMV-B) or wheat streak mosaic virus (WSMV). A 1 to 9 visualrating indicating the resistance to Corn Lethal Necrosis. A higher scoreindicates a higher resistance. Data are collected only when sufficientselection pressure exists in the experiment measured.

COMRST=COMMON RUST (Puccinia sorghi). A 1 to 9 visual rating indicatingthe resistance to Common Rust. A higher score indicates a higherresistance. Data are collected only when sufficient selection pressureexists in the experiment measured.

CP=PERCENT OF CRUDE PROTEIN. The percent of dry matter that is crudeprotein in chopped whole plant forage.

CROSS POLLINATION. A plant is cross pollinated if the pollen comes froma flower on a different plant from a different family or line. Crosspollination excludes sib and self pollination.

CROSS. As used herein, the term “cross” or “crossing” can refer to asimple X by Y cross, or the process of backcrossing, depending on thecontext.

D/D=DRYDOWN. This represents the relative rate at which a hybrid willreach acceptable harvest moisture compared to other hybrids on a 1 to 9rating scale. A high score indicates a hybrid that dries relatively fastwhile a low score indicates a hybrid that dries slowly.

DIPERS=DIPLODIA EAR MOLD SCORES (Diplodia maydis and Diplodiamacrospora). A 1 to 9 visual rating indicating the resistance toDiplodia Ear Mold. A higher score indicates a higher resistance. Dataare collected only when sufficient selection pressure exists in theexperiment measured.

DIPLOID PLANT PART. Refers to a plant part or cell that has the samediploid genotype as 32H20.

DIPROT=DIPLODIA STALK ROT SCORE. Score of stalk rot severity due toDiplodia (Diplodia maydis). Expressed as a 1 to 9 score with 9 beinghighly resistant. Data are collected only when sufficient selectionpressure exists in the experiment measured.

DM=PERCENT OF DRY MATTER. The percent of dry material in chopped wholeplant silage.

DRPEAR=DROPPED EARS. A measure of the number of dropped ears per plotand represents the percentage of plants that did not drop ears prior toharvest. Data are collected only when sufficient selection pressureexists in the experiment measured.

D/T=DROUGHT TOLERANCE. This represents a 1 to 9 rating for droughttolerance, and is based on data obtained under stress conditions. A highscore indicates good drought tolerance and a low score indicates poordrought tolerance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

EARHT=EAR HEIGHT. The ear height is a measure from the ground to thehighest placed developed ear node attachment and is measured incentimeters.

EARMLD=GENERAL EAR MOLD. Visual rating (1 to 9 score) where a 1 is verysusceptible and a 9 is very resistant. This is based on overall ratingfor ear mold of mature ears without determining the specific moldorganism, and may not be predictive for a specific ear mold. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

EARSZ=EAR SIZE. A 1 to 9 visual rating of ear size. The higher therating the larger the ear size.

EBTSTK=EARLY BRITTLE STALK. A count of the number of “snapped” plantsper plot following severe winds when the corn plant is experiencing veryrapid vegetative growth in the V5-V8 stage. Expressed as percent ofplants that did not snap. Data are collected only when sufficientselection pressure exists in the experiment measured.

ECB1 LF=EUROPEAN CORN BORER FIRST GENERATION LEAF FEEDING (Ostrinianubilalis). A 1 to 9 visual rating indicating the resistance topreflowering leaf feeding by first generation European Corn Borer. Ahigher score indicates a higher resistance. Data are collected only whensufficient selection pressure exists in the experiment measured.

ECB21T=EUROPEAN CORN BORER SECOND GENERATION INCHES OF TUNNELING(Ostrinia nubilalis). Average inches of tunneling per plant in thestalk. Data are collected only when sufficient selection pressure existsin the experiment measured.

ECB2SC=EUROPEAN CORN BORER SECOND GENERATION (Ostrinia nubilalis). A 1to 9 visual rating indicating post flowering degree of stalk breakageand other evidence of feeding by second generation European Corn Borer.A higher score indicates a higher resistance. Data are collected onlywhen sufficient selection pressure exists in the experiment measured.

ECBDPE=EUROPEAN CORN BORER DROPPED EARS (Ostrinia nubilalis). Droppedears due to European Corn Borer. Percentage of plants that did not dropears under second generation European Corn Borer infestation. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

ECBLSI=EUROPEAN CORN BORER LATE SEASON INTACT (Ostrinia nubilalis). A 1to 9 visual rating indicating late season intactness of the corn plantgiven damage (stalk breakage above and below the top ear) causedprimarily by 2^(nd) and/or 3^(rd) generation ECB larval feeding beforeharvest. A higher score is good and indicates more intact plants. Dataare collected only when sufficient selection pressure exists in theexperiment measured.

EGRWTH=EARLY GROWTH. This is a measure of the relative height and sizeof a corn seedling at the 2-4 leaf stage of growth. This is a visualrating (1 to 9), with 1 being weak or slow growth, 5 being averagegrowth and 9 being strong growth. Taller plants, wider leaves, moregreen mass and darker color constitute a higher score. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

ELITE INBRED. An inbred that contributed desirable qualities when usedto produce commercial hybrids. An elite inbred may also be used infurther breeding for the purpose of developing further improvedvarieties.

ERTLDG=EARLY ROOT LODGING. The percentage of plants that do not rootlodge prior to or around anthesis; plants that lean from the verticalaxis at an approximately 30 degree angle or greater would be counted asroot lodged. Data are collected only when sufficient selection pressureexists in the experiment measured.

ERTLPN=EARLY ROOT LODGING. An estimate of the percentage of plants thatdo not root lodge prior to or around anthesis; plants that lean from thevertical axis at an approximately 30 degree angle or greater would beconsidered as root lodged. Data are collected only when sufficientselection pressure exists in the experiment measured.

ERTLSC=EARLY ROOT LODGING SCORE. Score for severity of plants that leanfrom a vertical axis at an approximate 30 degree angle or greater whichtypically results from strong winds prior to or around floweringrecorded within 2 weeks of a wind event. Expressed as a 1 to 9 scorewith 9 being no lodging. Data are collected only when sufficientselection pressure exists in the experiment measured.

ESTCNT=EARLY STAND COUNT. This is a measure of the stand establishmentin the spring and represents the number of plants that emerge on a perplot basis for the inbred or hybrid.

EYESPT=EYE SPOT (Kabatiella zeae or Aureobasidium zeae). A 1 to 9 visualrating indicating the resistance to Eye Spot. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

FUSERS=FUSARIUM EAR ROT SCORE (Fusarium moniliforme or Fusariumsubglutinans). A 1 to 9 visual rating indicating the resistance toFusarium ear rot. A higher score indicates a higher resistance. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

GDU=Growing Degree Units. Using the Barger Heat Unit Theory, whichassumes that maize growth occurs in the temperature range 50 degreesF.-86 degrees F. and that temperatures outside this range slow downgrowth; the maximum daily heat unit accumulation is 36 and the minimumdaily heat unit accumulation is 0. The seasonal accumulation of GDU is amajor factor in determining maturity zones.

GDUSHD=GDU TO SHED. The number of growing degree units (GDUs) or heatunits required for an inbred line or hybrid to have approximately 50percent of the plants shedding pollen and is measured from the time ofplanting. Growing degree units are calculated by the Barger Method,where the heat units for a 24-hour period are:${GDU} = {\frac{\left( {{Max}.\quad{temp}.{+ {{Min}.\quad{temp}.}}} \right)}{2} - 50}$

The highest maximum temperature used is 86 degrees F. and the lowestminimum temperature used is 50 degrees F. For each inbred or hybrid ittakes a certain number of GDUs to reach various stages of plantdevelopment.

GDUSLK=GDU TO SILK. The number of growing degree units required for aninbred line or hybrid to have approximately 50 percent of the plantswith silk emergence from time of planting. Growing degree units arecalculated by the Barger Method as given in GDU SHD definition.

GENE SILENCING. The interruption or suppression of the expression of agene at the level of transcription or translation.

GENOTYPE. Refers to the genetic constitution of a cell or organism.

GIBERS=GIBBERELLA EAR ROT (PINK MOLD) (Gibberella zeae). A 1 to 9 visualrating indicating the resistance to Gibberella Ear Rot. A higher scoreindicates a higher resistance. Data are collected only when sufficientselection pressure exists in the experiment measured.

GIBROT=GIBBERELLA STALK ROT SCORE. Score of stalk rot severity due toGibberella (Gibberella zeae). Expressed as a 1 to 9 score with 9 beinghighly resistant. Data are collected only when sufficient selectionpressure exists in the experiment measured.

GLFSPT=GRAY LEAF SPOT (Cercospora zeae-maydis). A 1 to 9 visual ratingindicating the resistance to Gray Leaf Spot. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

GOSWLT=GOSS' WILT (Corynebacterium nebraskense). A 1 to 9 visual ratingindicating the resistance to Goss' Wilt. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

GRNAPP=GRAIN APPEARANCE. This is a 1 to 9 rating for the generalappearance of the shelled grain as it is harvested based on such factorsas the color of harvested grain, any mold on the grain, and any crackedgrain. High scores indicate good grain quality.

H/POP=YIELD AT HIGH DENSITY. Yield ability at relatively high plantdensities on a 1 to 9 relative rating system with a higher numberindicating the hybrid responds well to high plant densities for yieldrelative to other hybrids. A 1, 5, and 9 would represent very poor,average, and very good yield response, respectively, to increased plantdensity.

HAPLOID PLANT PART. Refers to a plant part or cell that has the samehaploid genotype as 32H20.

HCBLT=HELMINTHOSPORIUM CARBONUM LEAF BLIGHT (Helminthosporium carbonum).A 1 to 9 visual rating indicating the resistance to Helminthosporiuminfection. A higher score indicates a higher resistance. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

HD SMT=HEAD SMUT (Sphacelotheca reiliana). This score indicates thepercentage of plants not infected. Data are collected only whensufficient selection pressure exists in the experiment measured.

HSKCVR=HUSK COVER. A 1 to 9 score based on performance relative to keychecks, with a score of 1 indicating very short husks, tip of ear andkernels showing; 5 is intermediate coverage of the ear under mostconditions, sometimes with thin husk; and a 9 has husks extending andclosed beyond the tip of the ear. Scoring can best be done nearphysiological maturity stage or any time during dry down untilharvested.

INBRED. A line developed through inbreeding or doubled haploidy thatpreferably comprises homozygous alleles at about 95% or more of itsloci.

INC D/A=GROSS INCOME (DOLLARS PER ACRE). Relative income per acreassuming drying costs of two cents per point above 15.5 percent harvestmoisture and current market price per bushel.

INCOME/ACRE. Income advantage of hybrid to be patented over other hybridon per acre basis.

INC ADV=GROSS INCOME ADVANTAGE. Gross income advantage of variety #1over variety #2.

INTROGRESSION. The process of transferring genetic material from onegenotype to another.

KSZDCD=KERNEL SIZE DISCARD. The percent of discard seed; calculated asthe sum of discarded tip kernels and extra large kernels.

LINKAGE. Refers to a phenomenon wherein alleles on the same chromosometend to segregate together more often than expected by chance if theirtransmission was independent.

LINKAGE DISEQUILIBRIUM. Refers to a phenomenon wherein alleles tend toremain together in linkage groups when segregating from parents tooffspring, with a greater frequency than expected from their individualfrequencies.

LOCUS. A defined segment of DNA.

L/POP=YIELD AT LOW DENSITY. Yield ability at relatively low plantdensities on a 1 to 9 relative system with a higher number indicatingthe hybrid responds well to low plant densities for yield relative toother hybrids. A 1, 5, and 9 would represent very poor, average, andvery good yield response, respectively, to low plant density.

LRTLDG=LATE ROOT LODGING. The percentage of plants that do not rootlodge after anthesis through harvest; plants that lean from the verticalaxis at an approximately 30 degree angle or greater would be counted asroot lodged. Data are collected only when sufficient selection pressureexists in the experiment measured.

LRTLPN=LATE ROOT LODGING. An estimate of the percentage of plants thatdo not root lodge after anthesis through harvest; plants that lean fromthe vertical axis at an approximately 30 degree angle or greater wouldbe considered as root lodged. Data are collected only when sufficientselection pressure exists in the experiment measured.

LRTLSC=LATE ROOT LODGING SCORE. Score for severity of plants that leanfrom a vertical axis at an approximate 30 degree angle or greater whichtypically results from strong winds after flowering. Recorded prior toharvest when a root-lodging event has occurred. This lodging results inplants that are leaned or “lodged” over at the base of the plant and donot straighten or “goose-neck” back to a vertical position. Expressed asa 1 to 9 score with 9 being no lodging. Data are collected only whensufficient selection pressure exists in the experiment measured.

MDMCPX=MAIZE DWARF MOSAIC COMPLEX (MDMV=Maize Dwarf Mosaic Virus andMCDV=Maize Chlorotic Dwarf Virus). A 1 to 9 visual rating indicating theresistance to Maize Dwarf Mosaic Complex. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

MST=HARVEST MOISTURE. The moisture is the actual percentage moisture ofthe grain at harvest.

MSTADV=MOISTURE ADVANTAGE. The moisture advantage of variety #1 overvariety #2 as calculated by: MOISTURE of variety #2−MOISTURE of variety#1=MOISTURE ADVANTAGE of variety #1.

NEI DISTANCE. A quantitative measure of percent similarity between twolines. Nei's distance between lines A and B can be defined as1−(2*number alleles in common/(number alleles in A+number alleles in B).For example, if lines A and B are the same for 95 out of 100 alleles,the Nei distance would be 0.05. If lines A and B are the same for 98 outof 100 alleles, the Nei distance would be 0.02. Free software forcalculating Nei distance is available on the internet at multiplelocations such as, for example, at:evolution.genetics.washington.edu/phylip.html. See Nei, Proc Natl AcadSci, 76:5269-5273 (1979) which is incorporated by reference for thispurpose.

NLFBLT=NORTHERN LEAF BLIGHT (Helminthosporium turcicum or Exserohilumturcicum). A 1 to 9 visual rating indicating the resistance to NorthernLeaf Blight. A higher score indicates a higher resistance. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

OILT=GRAIN OIL. Absolute value of oil content of the kernel as predictedby Near-Infrared Transmittance and expressed as a percent of dry matter.

PEDIGREE DISTANCE. Relationship among generations based on theirancestral links as evidenced in pedigrees. May be measured by thedistance of the pedigree from a given starting point in the ancestry.

PERCENT IDENTITY. Percent identity as used herein refers to thecomparison of the alleles of two plants or lines as scored by matchingloci. Percent identity is determined by comparing a statisticallysignificant number of the loci of two plants or lines and scoring amatch when the same two alleles are present at the same loci for eachplant. For example, a percent identity of 90% between hybrid 32H20 andanother plant means that the two plants have the same two alleles at 90%of their loci.

PERCENT SIMILARITY. Percent similarity as used herein refers to thecomparison of the alleles of two plants or lines as scored by matchingalleles. Percent similarity is determined by comparing a statisticallysignificant number of the loci of two plants or lines and scoring oneallele match when the same allele is present at the same loci for eachplant and two allele matches when the same two alleles are present atthe same loci for each plant. A percent similarity of 90% between hybrid32H20 and another plant means that the two plants have 90% matchingalleles.

PLANT. As used herein, the term “plant” includes reference to animmature or mature whole plant, including a plant that has beendetasseled or from which seed or grain has been removed. Seed or embryothat will produce the plant is also considered to be the plant.

PLANT PARTS. As used herein, the term “plant parts” includes leaves,stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs,husks, stalks, root tips, anthers, pericarp, silk, tissue, cells and thelike.

PLTHT=PLANT HEIGHT. This is a measure of the height of the plant fromthe ground to the tip of the tassel in centimeters.

POLSC=POLLEN SCORE. A 1 to 9 visual rating indicating the amount ofpollen shed. The higher the score the more pollen shed.

POLWT=POLLEN WEIGHT. This is calculated by dry weight of tasselscollected as shedding commences minus dry weight from similar tasselsharvested after shedding is complete.

POP K/A=PLANT POPULATIONS. Measured as 1000 s per acre.

POP ADV=PLANT POPULATION ADVANTAGE. The plant population advantage ofvariety #1 over variety #2 as calculated by PLANT POPULATION of variety#2−PLANT POPULATION of variety #1=PLANT POPULATION ADVANTAGE of variety#1.

PRM=PREDICTED RELATIVE MATURITY. This trait, predicted relativematurity, is based on the harvest moisture of the grain. The relativematurity rating is based on a known set of checks and utilizes standardlinear regression analyses and is also referred to as the ComparativeRelative Maturity Rating System that is similar to the MinnesotaRelative Maturity Rating System.

PRMSHD=A relative measure of the growing degree units (GDU) required toreach 50% pollen shed. Relative values are predicted values from thelinear regression of observed GDU's on relative maturity of commercialchecks.

PROT=GRAIN PROTEIN. Absolute value of protein content of the kernel aspredicted by Near-Infrared Transmittance and expressed as a percent ofdry matter.

RESISTANCE. Synonymous with tolerance. The ability of a plant towithstand exposure to an insect, disease, herbicide or other condition.A resistant plant variety will have a level of resistance higher than acomparable wild-type variety.

RTLDG=ROOT LODGING. Root lodging is the percentage of plants that do notroot lodge; plants that lean from the vertical axis at an approximately30 degree angle or greater would be counted as root lodged. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

RTLADV=ROOT LODGING ADVANTAGE. The root lodging advantage of variety #1over variety #2. Data are collected only when sufficient selectionpressure exists in the experiment measured.

SCTGRN=SCATTER GRAIN. A 1 to 9 visual rating indicating the amount ofscatter grain (lack of pollination or kernel abortion) on the ear. Thehigher the score the less scatter grain.

SDGVGR=SEEDLING VIGOR. This is the visual rating (1 to 9) of the amountof vegetative growth after emergence at the seedling stage(approximately five leaves). A higher score indicates better vigor.

SEL IND=SELECTION INDEX. The selection index gives a single measure ofthe hybrid's worth based on information for up to five traits. A maizebreeder may utilize his or her own set of traits for the selectionindex. One of the traits that is almost always included is yield. Theselection index data presented in the tables represent the mean valueaveraged across testing stations.

SIL DMP=SILAGE DRY MATTER. The percent of dry material in chopped wholeplant silage.

SELF POLLINATION. A plant is self-pollinated if pollen from one floweris transferred to the same or another flower of the same plant.

SIB POLLINATION. A plant is sib-pollinated when individuals within thesame family or line are used for pollination.

SINGLE LOCUS CONVERSION TRAIT. A trait that can be introgressed into acorn line through introgression and/or transformation of a single locus.Examples of such single locus traits include mutant genes, transgenesand native traits finely mapped to a single locus. One or more singlelocus conversion traits may be introduced into a single corn line.

SLFBLT=SOUTHERN LEAF BLIGHT (Helminthosporium maydis or Bipolarismaydis). A 1 to 9 visual rating indicating the resistance to SouthernLeaf Blight. A higher score indicates a higher resistance. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

SOURST=SOUTHERN RUST (Puccinia polysora). A 1 to 9 visual ratingindicating the resistance to Southern Rust. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

STAGRN=STAY GREEN. Stay green is the measure of plant health near thetime of black layer formation (physiological maturity). A high scoreindicates better late-season plant health.

STARCH=PERCENT OF STARCH. The percent of dry matter that is starch inchopped whole plant forage.

STDADV=STALK STANDING ADVANTAGE. The advantage of variety #1 overvariety #2 for the trait STK CNT.

STKCNT=NUMBER OF PLANTS. This is the final stand or number of plants perplot.

STKLDG=STALK LODGING REGULAR. This is the percentage of plants that didnot stalk lodge (stalk breakage) at regular harvest (when grain moistureis between about 20 and 30%) as measured by either natural lodging orpushing the stalks and determining the percentage of plants that breakbelow the ear. Data are collected only when sufficient selectionpressure exists in the experiment measured.

STKLDS=STALK LODGING SCORE. A plant is considered as stalk lodged if thestalk is broken or crimped between the ear and the ground. This can becaused by any or a combination of the following: strong winds late inthe season, disease pressure within the stalks, ECB damage orgenetically weak stalks. This trait should be taken just prior to or atharvest. Expressed on a 1 to 9 scale with 9 being no lodging. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

STLLPN=LATE STALK LODGING. This is the percent of plants that did notstalk lodge (stalk breakage or crimping) at or around late seasonharvest (when grain moisture is below 20%) as measured by either naturallodging or pushing the stalks and determining the percentage of plantsthat break or crimp below the ear. Data are collected only whensufficient selection pressure exists in the experiment measured.

STLPCN=STALK LODGING REGULAR. This is an estimate of the percentage ofplants that did not stalk lodge (stalk breakage) at regular harvest(when grain moisture is between about 20 and 30%) as measured by eithernatural lodging or pushing the stalks and determining the percentage ofplants that break below the ear. Data are collected only when sufficientselection pressure exists in the experiment measured.

STRT=GRAIN STARCH. Absolute value of starch content of the kernel aspredicted by Near-Infrared Transmittance and expressed as a percent ofdry matter.

STWWLT=Stewart's Wilt (Erwinia stewartii). A 1 to 9 visual ratingindicating the resistance to Stewart's Wilt. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

TASBLS=TASSEL BLAST. A 1 to 9 visual rating was used to measure thedegree of blasting (necrosis due to heat stress) of the tassel at thetime of flowering. A 1 would indicate a very high level of blasting attime of flowering, while a 9 would have no tassel blasting. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

TASSZ=TASSEL SIZE. A 1 to 9 visual rating was used to indicate therelative size of the tassel. The higher the rating the larger thetassel.

TAS WT=TASSEL WEIGHT. This is the average weight of a tassel (grams)just prior to pollen shed.

TDM/HA=TOTAL DRY MATTER PER HECTARE. Yield of total dry plant materialin metric tons per hectare.

TEXEAR=EAR TEXTURE. A 1 to 9 visual rating was used to indicate therelative hardness (smoothness of crown) of mature grain. A 1 would bevery soft (extreme dent) while a 9 would be very hard (flinty or verysmooth crown).

TILLER=TILLERS. A count of the number of tillers per plot that couldpossibly shed pollen was taken. Data are given as a percentage oftillers: number of tillers per plot divided by number of plants perplot.

TST WT=TEST WEIGHT (UNADJUSTED). The measure of the weight of the grain(pounds) for a given volume (bushel).

TSWADV=TEST WEIGHT ADVANTAGE. The test weight advantage of variety #1over variety #2.

WIN M %=PERCENT MOISTURE WINS.

WIN Y %=PERCENT YIELD WINS.

YIELD BU/A=YIELD (BUSHELS/ACRE). Yield of the grain at harvest by weightor volume (bushels) per unit area (acre) adjusted to 15% moisture.

YLDADV=YIELD ADVANTAGE. The yield advantage of variety #1 over variety#2 as calculated by: YIELD of variety #1−YIELD variety #2=YIELDADVANTAGE of variety #1.

YLDSC=YIELD SCORE. A 1 to 9 visual rating was used to give a relativerating for yield based on plot ear piles. The higher the rating thegreater visual yield appearance.

Definitions for Area of Adaptability

When referring to area of adaptability, such term is used to describethe location with the environmental conditions that would be well suitedfor this maize line. Area of adaptability is based on a number offactors, for example: days to maturity, insect resistance, diseaseresistance, and drought resistance. Area of adaptability does notindicate that the maize line will grow in every location within the areaof adaptability or that it will not grow outside the area.

Central Corn Belt: Iowa, Illinois, Indiana

Drylands: non-irrigated areas of North Dakota, South Dakota, Nebraska,Kansas, Colorado and Oklahoma

Eastern U.S.: Ohio, Pennsylvania, Delaware, Maryland, Virginia, and WestVirginia

North central U.S.: Minnesota and Wisconsin

Northeast: Michigan, New York, Vermont, and Ontario and Quebec Canada

Northwest U.S.: North Dakota, South Dakota, Wyoming, Washington, Oregon,Montana, Utah, and Idaho

South central U.S.: Missouri, Tennessee, Kentucky, Arkansas

Southeast U.S.: North Carolina, South Carolina, Georgia, Florida,Alabama, Mississippi, and Louisiana

Southwest U.S.: Texas, Oklahoma, New Mexico, Arizona

Western U.S.: Nebraska, Kansas, Colorado, and California

Maritime Europe: Northern France, Germany, Belgium, Netherlands andAustria

DETAILED DESCRIPTION OF THE INVENTION

All tables discussed in the Detailed Description of the Invention andFurther Embodiments section can found at the end of the section.

Maize hybrids need to be highly homogeneous, heterozygous andreproducible to be useful as commercial hybrids. There are manyanalytical methods available to determine the heterozygous nature andthe identity of these lines.

The oldest and most traditional method of analysis is the observation ofphenotypic traits. The data is usually collected in field experimentsover the life of the maize plants to be examined. Phenotypiccharacteristics most often observed are for traits associated with plantmorphology, ear and kernel morphology, insect and disease resistance,maturity, and yield.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. A plant's genotype can be used to identify plants of thesame variety or a related variety. For example, the genotype can be usedto determine the pedigree of a plant. There are many laboratory-basedtechniques available for the analysis, comparison and characterizationof plant genotype; among these are Isozyme Electrophoresis, RestrictionFragment Length Polymorphisms (RFLPs), Randomly Amplified PolymorphicDNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Amplified Fragment Length Polymorphisms (AFLPs), SimpleSequence Repeats (SSRs) which are also referred to as Microsatellites,and Single Nucleotide Polymorphisms (SNPs).

Isozyme Electrophoresis and RFLPs as discussed in Lee, M., “Inbred Linesof Maize and Their Molecular Markers,” The Maize Handbook,(Springer-Verlag, New York, Inc. 1994, at 423-432) incorporated hereinby reference, have been widely used to determine genetic composition.Isozyme Electrophoresis has a relatively low number of available markersand a low number of allelic variants. RFLPs allow more discriminationbecause they have a higher degree of allelic variation in maize and alarger number of markers can be found. Both of these methods have beeneclipsed by SSRs as discussed in Smith et al., “An evaluation of theutility of SSR loci as molecular markers in maize (Zea mays L.):comparisons with data from RFLPs and pedigree”, Theoretical and AppliedGenetics (1997) vol. 95 at 163-173 and by Pejic et al., “Comparativeanalysis of genetic similarity among maize inbreds detected by RFLPs,RAPDs, SSRs, and AFLPs,” Theoretical and Applied Genetics (1998) at1248-1255 incorporated herein by reference. SSR technology is moreefficient and practical to use than RFLPs; more marker loci can beroutinely used and more alleles per marker locus can be found using SSRsin comparison to RFLPs. Single Nucleotide Polymorphisms may also be usedto identify the unique genetic composition of the invention and progenylines retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Maize DNA molecular marker linkage maps have been rapidly constructedand widely implemented in genetic studies. One such study is describedin Boppenmaier, et al., “Comparisons among strains of inbreds forRFLPs”, Maize Genetics Cooperative Newsletter, 65:1991, pg. 90, isincorporated herein by reference.

Pioneer Brand Hybrid 32H20 is characterized by excellent yield, goodstandibility and moderate plant and ear heights. This hybrid has goodresistance to Grey Leaf Spot and Goss's Wilt and also has goodstaygreen. The hybrid is particularly suited to the Southcentral U.S.and Europe.

Pioneer Brand Hybrid 32H20 is a single cross, yellow endosperm,flint-dent maize hybrid. Hybrid 32H20 has a relative maturity ofapproximately 115 based on the Comparative Relative Maturity RatingSystem for harvest moisture of grain.

The hybrid has shown uniformity and stability within the limits ofenvironmental influence for all the traits as described in the VarietyDescription Information (Table 1, found at the end of the section). Theinbred parents of this hybrid have been self-pollinated and ear-rowed asufficient number of generations with careful attention paid touniformity of plant type to ensure the homozygosity and phenotypicstability necessary for use in commercial hybrid seed production. Theline has been increased both by hand and in isolated fields withcontinued observation for uniformity. No variant traits have beenobserved or are expected in 32H20.

Hybrid 32H20 can be reproduced by planting seeds of the inbred parentlines, growing the resulting maize plants under cross pollinatingconditions, and harvesting the resulting seed using techniques familiarto the agricultural arts.

Comparisons for Pioneer Hybrid 32H20

A breeder uses various methods to help determine which plants should beselected from segregating populations and ultimately which inbred lineswill be used to develop hybrids for commercialization. In addition toknowledge of the germplasm and plant genetics, a part of the selectionprocess is dependent on experimental design coupled with the use ofstatistical analysis. Experimental design and statistical analysis areused to help determine which plants, which family of plants, and finallywhich inbred lines and hybrid combinations are significantly better ordifferent for one or more traits of interest. Experimental designmethods are used to assess error so that differences between two inbredlines or two hybrid lines can be more accurately evaluated. Statisticalanalysis includes the calculation of mean values, determination of thestatistical significance of the sources of variation, and thecalculation of the appropriate variance components. Either a five or aone percent significance level is customarily used to determine whethera difference that occurs for a given trait is real or due to theenvironment or experimental error. One of ordinary skill in the art ofplant breeding would know how to evaluate the traits of two plantvarieties to determine if there is no significant difference between thetwo traits expressed by those varieties. For example, see Fehr, Walt,Principles of Cultivar Development, pages 261-286 (1987). Mean traitvalues may be used to determine whether trait differences aresignificant. Trait values should preferably be measured on plants grownunder the same environmental conditions, and environmental conditionsshould be appropriate for the traits or traits being evaluated.Sufficient selection pressure should be present for optimum measurementof traits of interest such as herbicide, insect or disease resistance.Similarly, an introgressed trait conversion of 32H20 for resistance,such as herbicide resistance, should not be compared to 32H20 in thepresence of the herbicide when comparing non-resistance related traitssuch as plant height and yield.

In Table 2 (Table 2, found at the end of the section), data from traitsand characteristics of hybrid 32H20 per se are given and compared toother maize hybrids. The following are the results of these comparisons.The results in Table 2 show hybrid 32H20 has significantly differenttraits compared to other maize hybrids.

Comparisons of characteristics for Pioneer Brand Hybrid 32H20 were madeagainst Hybrid 33Y45 and Hybrid 32K22.

Table 2A compares Pioneer Brand Hybrid 32H20 and Hybrid 33Y45, a hybridwith a similar area of adaptation. The results show Hybrid 32H20 differssignificantly in a number of traits including plant height and earheight when compared to Hybrid 33Y45. Table 2B compares Pioneer BrandHybrid 32H20 and Hybrid 32K22, a hybrid with a similar area ofadaptation. The results show that Hybrid 32K22 differs significantly ina number of traits including ear height, stay green and resistance toGray Leaf Spot.

Further Embodiments of the Invention

This invention also is directed to methods for producing a maize plantby crossing a first parent maize plant with a second parent maize plantwherein either the first or second parent maize plant is Pioneer Brandhybrid 32H20. In one embodiment the parent hybrid maize plant 32H20 willbe crossed with another maize plant, sibbed, or selfed, to generate aninbred which may be used in the development of additional plants. Inanother embodiment, double haploid methods may be used to generate aninbred plant. Further, this invention is directed to methods forproducing a hybrid 32H20-progeny maize plant by crossing hybrid maizeplant 32H20 with itself or a second maize plant and growing the progenyseed, and repeating the crossing and growing steps with the hybrid maize32H20-progeny plant from 1 to 2 times, 1 to 3 times, 1 to 4 times, or 1to 5 times. Thus, any such methods using the hybrid maize plant 32H20are part of this invention: selfing, sibbing, backcrosses, hybridproduction, crosses to populations, and the like.

All plants produced by the use of the methods described herein and thatretain the unique genetic or trait combination of hybrid maize plant32H20 as a parent are within the scope of this invention, includingplants derived from hybrid maize plant 32H20. Progeny of the breedingmethods described herein may be characterized in any number of ways,such as by traits retained in the progeny, pedigree and/or molecularmarkers. Combinations of these methods of characterization may be used.This includes varieties essentially derived from variety 32H20.Breeder's of ordinary skill in the art have developed the concept of an“essentially derived variety”, which is defined in 7 U.S.C. § 2104(a)(3)of the Plant Variety Protection Act and is hereby incorporated byreference. Varieties and plants that are essentially derived from 32H20are within the scope of the invention. This also includes progeny plantand parts thereof with at least one ancestor that is hybrid maize plant32H20 and more specifically where the pedigree of this progeny includes1, 2, 3, 4, and/or 5 or cross pollinations to a maize plant 32H20, or aplant that has 32H20 as a progenitor. Pedigree is a method used bybreeders of ordinary skill in the art to describe the varieties.Varieties that are more closely related by pedigree are likely to sharecommon genotypes and combinations of phenotypic characteristics. Allbreeders of ordinary skill in the art maintain pedigree records of theirbreeding programs. These pedigree records contain a detailed descriptionof the breeding process, including a listing of all parental lines usedin the breeding process and information on how such line was used. Thus,a breeder of ordinary skill in the art would know if 32H20 were used inthe development of a progeny line, and would also know how many breedingcrosses to a line other than 32H20 or line with 32H20 as a parent orother progenitor were made in the development of any progeny line. Aprogeny line so developed may then be used in crosses with other,different, maize inbreds to produce first generation (F₁) maize hybridseeds and plants with superior characteristics.

Specific methods and products produced using hybrid maize plant in plantbreeding are encompassed within the scope of the invention listed above.One such embodiment is the process of crossing hybrid maize plant 32H20with itself to form a homozygous inbred parent line. Hybrid 32H20 wouldbe sib or self pollinated to form a population of progeny plants. Thepopulation of progeny plants produced by this method is also anembodiment of the invention. This first population of progeny plantswill have received all of its alleles from hybrid maize plant 32H20. Theinbreeding process results in homozygous loci being generated and isrepeated until the plant is homozygous at substantially every loci andbecomes an inbred line. Once this is accomplished the inbred line may beused in crosses with other inbred lines, including but not limited toinbred parent lines disclosed herein to generate a first generation ofF1 hybrid plants. One of ordinary skill in the art can utilize breedernotebooks, or molecular methods to identify a particular hybrid plantproduced using an inbred line derived from maize hybrid plant 32H20, inaddition to comparing traits. Any such individual inbred plant is alsoencompassed by this invention.

These embodiments also include use of these methods with transgenic orbackcross conversions of maize hybrid plant 32H20. Another suchembodiment is a method of developing a line genetically similar tohybrid maize plant 32H20 in breeding that involves the repeatedbackcrossing of an inbred parent of, or an inbred line derived from,hybrid maize plant 32H20. Using backcrossing methods, or even the tissueculture and transgenic methods described herein, the backcrossconversion methods described herein, or other breeding methods known toone of ordinary skill in the art, one can develop individual plants,plant cells, and populations of plants that retain at least 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 99.5% genetic similarity or identity to maize hybridplant 32H20, as measured by either percent identity or percentsimilarity. The percentage of the genetics retained in the progeny maybe measured by either pedigree analysis or through the use of genetictechniques such as molecular markers or electrophoresis. In pedigreeanalysis, on average 50% of the starting germplasm would be expected tobe passed to the progeny line after one cross to a different line, 25%after another cross to another line, and so on. Actual geneticcontribution may be much higher than the genetic contribution expectedby pedigree, especially if molecular markers are used in selection.Molecular markers could also be used to confirm and/or determine thepedigree of the progeny line. The inbred parent would then be crossed toa second inbred parent of or derived from hybrid maize plant 32H20 tocreate hybrid maize plant 32H20 with additional beneficial traits suchas transgenes or backcross conversions.

One method for producing a line derived from hybrid maize plant is asfollows. One of ordinary skill in the art would obtain hybrid maizeplant 32H20 and cross it with another variety of maize, such as an eliteinbred variety. The F1 seed derived from this cross would be grown toform a population. The nuclear genome of the F1 would be made-up of 50%of hybrid maize plant 32H20 and 50% of the other elite variety. The F1seed would be grown and allowed to self, thereby forming F2 seed. Onaverage the F2 seed nuclear genome would have derived 50% of its allelesfrom the parent hybrid plant 32H20 and 50% from the other maize variety,but many individual plants from the population would have a greaterpercentage of their alleles derived from, the parent maize hybrid plant(Wang J. and R. Bernardo, 2000, Crop Sci. 40:659-665 and Bernardo, R.and A. L. Kahler, 2001, Theor. Appl. Genet 102:986-992). Molecularmarkers of 32H20, or its parents identified from routine screening ofthe deposited samples herein could be used to select and retain thoselines with high similarity to 32H20. The F2 seed would be grown andselection of plants would be made based on visual observation, markersand/or measurement of traits. The traits used for selection may be any32H20 trait described in this specification, including the hybrid maizeplant 32H20 traits of high yield, good staygreen, resistance to GreyLeaf Spot and Goss's Wilt, and particularly suited to the Southcentralareas of the United States, and Europe. Such traits may also be the goodgeneral or specific combining ability of 32H20. The 32H20 progeny plantsthat exhibit one or more of the desired 32H20 traits, such as thoselisted above, would be selected and each plant would be harvestedseparately. This F3 seed from each plant would be grown in individualrows and allowed to self. Then selected rows or plants from the rowswould be harvested individually. The selections would again be based onvisual observation, markers and/or measurements for desirable traits ofthe plants, such as one or more of the desirable 32H20 traits listedherein.

The process of growing and selection would be repeated any number oftimes until a 32H20 progeny plant is obtained. The 32H20 progeny inbredplant would contain desirable traits derived from hybrid plant 32H20.The resulting progeny line would benefit from the efforts of theinventor(s), and would not have existed but for the inventor(s) work increating 32H20. Another embodiment of the invention is a 32H20 progenyplant that has received the desirable 32H20 traits listed above throughthe use of 32H20, which traits were not exhibited by other plants usedin the breeding process.

The previous example can be modified in numerous ways, for instanceselection may or may not occur at every selfing generation, the hybridmay immediately be selfed without crossing to another plant, selectionmay occur before or after the actual self-pollination process occurs, orindividual selections may be made by harvesting individual ears, plants,rows or plots at any point during the breeding process described. Inaddition, double haploid breeding methods may be used at any step in theprocess. The population of plants produced at each and any cycle ofbreeding is also an embodiment of the invention, and on average eachsuch population would predictably consist of plants containingapproximately 50% of its genes from inbred parents of maize hybrid 32H20in the first breeding cycle, 25% of its genes from inbred parents ofmaize hybrid 32H20 in the second breeding cycle, 12.5% of its genes frominbred parents of maize hybrid 32H20 in the third breeding cycle, 6.25%in the fourth breeding cycle, 3.125% in the fifth breeding cycle, and soon. In each case the use of 32H20 provides a substantial benefit. Thelinkage groups of 32H20 would be retained in the progeny lines, andsince current estimates of the maize genome size is about 50,000-80,000genes (Xiaowu, Gai et al., Nucleic Acids Research, 2000, Vol. 28, No. 1,94-96), in addition to non-coding DNA that impacts gene expression, itprovides a significant advantage to use 32H20 as starting material toproduce a line that retains desired genetics or traits of 32H20.

Another embodiment of this invention is the method of obtaining asubstantially homozygous 32H20 progeny plant by obtaining a seed fromthe cross of 32H20 and another maize plant and applying double haploidmethods to the F1 seed or F1 plant or to any successive filialgeneration. Such methods substantially decrease the number ofgenerations required to produce an inbred with similar genetics orcharacteristics to 32H20.

A further embodiment of the invention is a backcross conversion of 32H20obtained by crossing inbred parent plants of hybrid maize plant 32H20,which comprise the backcross conversion. For a dominant or additivetrait at least one of the inbred parents would include a backcrossconversion in its genome. For a recessive trait, each parent wouldinclude the backcross conversion in its genome. In each case theresultant hybrid maize plant 32H20 obtained from the cross of theparents includes a backcross conversion or transgene.

32H20 represents a new base genetic line into which a new locus or traitmay be introgressed. Direct transformation and backcrossing representtwo important methods that can be used to accomplish such anintrogression. A backcross conversion of 32H20 occurs when DNA sequencesare introduced through backcrossing (Hallauer et al., 1988), with aparent of 32H20 utilized as the recurrent parent. Both naturallyoccurring and transgenic DNA sequences may be introduced throughbackcrossing techniques. A backcross conversion may produce a plant witha trait or locus conversion in at least one or more backcrosses,including at least 2 crosses, at least 3 crosses, at least 4 crosses, atleast 5 crosses and the like. Molecular marker assisted breeding orselection may be utilized to reduce the number of backcrosses necessaryto achieve the backcross conversion. For example, see Openshaw, S. J. etal., Marker-assisted Selection in Backcross Breeding. In: ProceedingsSymposium of the Analysis of Molecular Data, August 1994, Crop ScienceSociety of America, Corvallis, Oreg., where it is demonstrated that abackcross conversion can be made in as few as two backcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes as vs.unlinked genes), the level of expression of the trait, the type ofinheritance (cytoplasmic or nuclear) and the types of parents includedin the cross. It is understood by those of ordinary skill in the artthat for single gene traits that are relatively easy to classify, thebackcross method is effective and relatively easy to manage. (SeeHallauer et al. in Corn and Corn Improvement, Sprague and Dudley, ThirdEd. 1998). Desired traits that may be transferred through single locustrait conversion include, but are not limited to, waxy starch, sterility(nuclear and cytoplasmic), fertility restoration, grain color (white),drought resistance enhanced nitrogen utilization efficiency, alterednitrogen responsiveness, altered fatty acid profile, disease resistance(bacterial, fungal or viral), insect resistance, herbicide resistanceand yield enhancements. In addition, an introgression site itself, suchas an FRT site, Lox site or other site specific integration site, may beinserted by backcrossing and utilized for direct insertion of one ormore genes of interest into a specific plant variety. The trait ofinterest is transferred from the donor parent to the recurrent parent,in this case, an inbred parent of the maize plant disclosed herein. Insome embodiments of the invention, the number of loci that may bebackcrossed into 32H20 is at least 1, 2, 3, 4, or 5 and/or no more than6, 5, 4, 3, or 2. A single loci may contain several transgenes, such asa transgene for disease resistance that, in the same expression vector,also contains a transgene for herbicide resistance. The gene forherbicide resistance may be used as a selectable marker and/or as aphenotypic trait. A single locus trait conversion of site specificintegration system allows for the integration of multiple genes at theconverted loci. Further, SSI and FRT technologies known to those ofskill in the art in the art may result in multiple gene introgressionsat a single loci.

The backcross conversion may result from either the transfer of adominant allele or a recessive allele. Selection of progeny containingthe trait of interest is accomplished by direct selection for a traitassociated with a dominant allele. Transgenes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele, such as the waxy starchcharacteristic, requires growing and selfing the first backcrossgeneration to determine which plants carry the recessive alleles.Recessive traits may require additional progeny testing in successivebackcross generations to determine the presence of the locus ofinterest. The last backcross generation is usually selfed to give purebreeding progeny for the gene(s) being transferred, although a backcrossconversion with a stably introgressed trait may also be maintained byfurther backcrossing to the recurrent parent with selection for theconverted trait.

Along with selection for the trait of interest, progeny are selected forthe phenotype of the recurrent parent. While occasionally additionalpolynucleotide sequences or genes may be transferred along with thebackcross conversion, the backcross conversion line “fits into the samehybrid combination as the recurrent parent inbred line and contributesthe effect of the additional gene added through the backcross.” SeePoehlman et al. (1995, page 334). A progeny comprising at least 95%,96%, 97%, 98%, 99%, 99.5% and 99.9% genetic identity to hybrid 32H20 andcomprising the backcross conversion trait or traits of interest, isconsidered to be a backcross conversion of hybrid 32H20. It has beenproposed that in general there should be at least four backcrosses whenit is important that the recovered lines be essentially identical to therecurrent parent except for the characteristic being transferred (Fehr1987, Principles of Cultivar Development). However, as noted above, thenumber of backcrosses necessary can be reduced with the use of molecularmarkers. Other factors, such as a genetically similar donor parent, mayalso reduce the number of backcrosses necessary.

Hybrid seed production requires elimination or inactivation of pollenproduced by the female inbred parent. Incomplete removal or inactivationof the pollen provides the potential for self-pollination. A reliablemethod of controlling male fertility in plants offers the opportunityfor improved seed production. There are several ways in which a maizeplant can be manipulated so that it is male sterile. These include useof manual or mechanical emasculation (or detasseling), use of one ormore genetic factors that confer male sterility, including cytoplasmicgenetic and/or nuclear genetic male sterility, use of gametocides andthe like. All of such embodiments are within the scope of the presentclaims. The term manipulated to be male sterile refers to the use of anyavailable techniques to produce a male sterile version of maize line32H20. The male sterility may be either partial or complete malesterility.

Hybrid maize seed is often produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twomaize inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Provided that there issufficient isolation from sources of foreign maize pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

Such embodiments are also within the scope of the present claims. Thisinvention includes hybrid maize seed of 32H20 and the hybrid maize plantproduced therefrom. The foregoing was set forth by way of example and isnot intended to limit the scope of the invention.

This invention is also directed to the use of hybrid maize plant 32H20in tissue culture. As used herein, the term “tissue culture” includesplant protoplasts, plant cell tissue culture, cultured microspores,plant calli, plant clumps, and the like. As used herein, phrases such as“growing the seed” or “grown from the seed” include embryo rescue,isolation of cells from seed for use in tissue culture, as well astraditional growing methods.

Duncan, Williams, Zehr, and Widholm, Planta, (1985) 165:322-332 reflectsthat 97% of the plants cultured which produced callus were capable ofplant regeneration. Subsequent experiments with both inbreds and hybridsproduced 91% regenerable callus which produced plants. In a furtherstudy in 1988, Songstad, Duncan & Widholm in Plant Cell Reports (1988),7:262-265 reports several media additions which enhance regenerabilityof callus of two inbred lines. Other published reports also indicatedthat “nontraditional” tissues are capable of producing somaticembryogenesis and plant regeneration. K. P. Rao, et al., Maize GeneticsCooperation Newsletter, 60:64-65 (1986), refers to somatic embryogenesisfrom glume callus cultures and B. V. Conger, et al., Plant Cell Reports,6:345-347 (1987) indicates somatic embryogenesis from the tissuecultures of maize leaf segments. Thus, it is clear from the literaturethat the state of the art is such that these methods of obtaining plantsare, and were, “conventional” in the sense that they are routinely usedand have a very high rate of success.

Tissue culture of maize, including tassel/anther culture, is describedin U.S. Application 2002/0062506A1 and European Patent Application,Publication EPO160,390, each of which are incorporated herein byreference for this purpose. Maize tissue culture procedures are alsodescribed in Green and Rhodes, “Plant Regeneration in Tissue Culture ofMaize,” Maize for Biological Research (Plant Molecular BiologyAssociation, Charlottesville, Va. 1982, at 367-372) and in Duncan, etal., “The Production of Callus Capable of Plant Regeneration fromImmature Embryos of Numerous Zea Mays Genotypes,” 165 Planta 322-332(1985). Thus, another aspect of this invention is to provide cells whichupon growth and differentiation produce maize plants having the genotypeand/or physiological and morphological characteristics of hybrid maizeplant 32H20.

The utility of hybrid maize plant 32H20 also extends to crosses withother species. Commonly, suitable species will be of the familyGraminaceae, and especially of the genera Zea, Tripsacum, Coix,Schlerachne, Polytoca, Chionachne, and Trilobachne, of the tribeMaydeae. Potentially suitable for crosses with 32H20 may be the variousvarieties of grain sorghum, Sorghum bicolor (L.) Moench.

Transformation of Maize

The advent of new molecular biological techniques has allowed theisolation and characterization of genetic elements with specificfunctions, such as encoding specific protein products. Scientists in thefield of plant biology developed a strong interest in engineering thegenome of plants to contain and express foreign genetic elements, oradditional, or modified versions of native or endogenous geneticelements in order to alter the traits of a plant in a specific manner.Any DNA sequences, whether from a different species or from the samespecies, which are inserted into the genome using transformation arereferred to herein collectively as “transgenes”. In some embodiments ofthe invention, a transformed variant of 32H20 may contain at least onetransgene but could contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10and/or no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.Over the last fifteen to twenty years several methods for producingtransgenic plants have been developed, and the present invention alsorelates to transformed versions of the claimed hybrid 32H20 as well ascombinations thereof.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88 and Armstrong, “The First Decade of Maize Transformation: A Reviewand Future Perspective” (Maydica 44:101-109, 1999). In addition,expression vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available. See, forexample, Gruber et al., “Vectors for Plant Transformation” in Methods inPlant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J.E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.

The most prevalent types of plant transformation involve theconstruction of an expression vector. Such a vector comprises a DNAsequence that contains a gene under the control of or operatively linkedto a regulatory element, for example a promoter. The vector may containone or more genes and one or more regulatory elements.

A genetic trait which has been engineered into the genome of aparticular maize plant using transformation techniques, could be movedinto the genome of another line using traditional breeding techniquesthat are well known in the plant breeding arts. These lines can then becrossed to generate a hybrid maize plant such as hybrid maize plant32H20 which comprises a transgene. For example, a backcrossing approachis commonly used to move a transgene from a transformed maize plant toan elite inbred line, and the resulting progeny would then comprise thetransgene(s). Also, if an inbred line was used for the transformationthen the transgenic plants could be crossed to a different inbred inorder to produce a transgenic hybrid maize plant. As used herein,“crossing” can refer to a simple X by Y cross, or the process ofbackcrossing, depending on the context.

Various genetic elements can be introduced into the plant genome usingtransformation. These elements include, but are not limited to genes;coding sequences; inducible, constitutive, and tissue specificpromoters; enhancing sequences; and signal and targeting sequences. Forexample, see the traits, genes and transformation methods listed in U.S.Pat. Nos. 6,118,055 and 6,284,953, which are herein incorporated byreference. In addition, transformability of a line can be increased byintrogressing the trait of high transformability from another line knownto have high transformability, such as Hi-II. See U.S. PatentApplication Publication US2004/0016030 (2004).

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants that areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114: 92-6(1981).

A genetic map can be generated, primarily via conventional RestrictionFragment Length Polymorphisms (RFLP), Polymerase Chain Reaction (PCR)analysis, Simple Sequence Repeats (SSR) and Single NucleotidePolymorphisms (SNP) that identifies the approximate chromosomal locationof the integrated DNA molecule. For exemplary methodologies in thisregard, see Glick and Thompson, Methods In Plant Molecular Biology AndBiotechnology, 269-284 (CRC Press, Boca Raton, 1993).

Wang et al. discuss “Large Scale Identification, Mapping and Genotypingof Single-Nucleotide Polymorphisms in the Human Genome”, Science,280:1077-1082, 1998, and similar capabilities are available for the corngenome. Map information concerning chromosomal location is useful forproprietary protection of a subject transgenic plant. If unauthorizedpropagation is undertaken and crosses made with other germplasm, the mapof the integration region can be compared to similar maps for suspectplants to determine if the latter have a common parentage with thesubject plant. Map comparisons would involve hybridizations, RFLP, PCR,SSR and sequencing, all of which are conventional techniques. SNPs mayalso be used alone or in combination with other techniques.

Likewise, by means of the present invention, plants can be geneticallyengineered to express various phenotypes of agronomic interest. Throughthe transformation of maize the expression of genes can be altered toenhance disease resistance, insect resistance, herbicide resistance,agronomic traits, grain quality and other traits. Transformation canalso be used to insert DNA sequences which control or help controlmale-sterility. DNA sequences native to maize as well as non-native DNAsequences can be transformed into maize and used to alter levels ofnative or non-native proteins. Various promoters, targeting sequences,enhancing sequences, and other DNA sequences can be inserted into themaize genome for the purpose of altering the expression of proteins.Reduction of the activity of specific genes (also known as genesilencing, or gene suppression) is desirable for several aspects ofgenetic engineering in plants.

Many techniques for gene silencing are well known to one of skill in theart, including but not limited to knock-outs (such as by insertion of atransposable element such as mu (Vicki Chandler, The Maize Handbook ch.118 (Springer-Verlag 1994) or other genetic elements such as a FRT, Loxor other site specific integration site, antisense technology (see,e.g., Sheehy et al. (1988) PNAS USA 85:8805-8809; and U.S. Pat. Nos.5,107,065; 5,453,566; and 5,759,829); co-suppression (e.g., Taylor(1997) Plant Cell 9:1245; Jorgensen (1990) Trends Biotech.8(12):340-344; Flavell (1994) PNAS USA 91:3490-3496; Finnegan et al.(1994) Bio/Technology 12: 883-888; and Neuhuber et al. (1994) Mol. Gen.Genet. 244:230-241); RNA interference (Napoli et al. (1990) Plant Cell2:279-289; U.S. Pat. No. 5,034,323; Sharp (1999) Genes Dev. 13:139-141;Zamore et al. (2000) Cell 101:25-33; and Montgomery et al. (1998) PNASUSA 95:15502-15507), virus-induced gene silencing (Burton, et al. (2000)Plant Cell 12:691-705; and Baulcombe (1999) Curr. Op. Plant Bio.2:109-113); target-RNA-specific ribozymes (Haseloff et al. (1988) Nature334: 585-591); hairpin structures (Smith et al. (2000) Nature407:319-320; WO 99/53050; and WO 98/53083); MicroRNA (Aukerman & Sakai(2003) Plant Cell 15:2730-2741); ribozymes (Steinecke et al. (1992) EMBOJ. 11:1525; and Perriman et al. (1993) Antisense Res. Dev. 3:253);oligonucleotide mediated targeted modification (e.g., WO 03/076574 andWO 99/25853); Zn-finger targeted molecules (e.g., WO 01/52620; WO03/048345; and WO 00/42219); and other methods or combinations of theabove methods known to those of skill in the art.

Exemplary nucleotide sequences that may be altered by geneticengineering include, but are not limited to, those categorized below.

1. Transgenes That Confer Resistance To Insects or Disease And ThatEncode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266: 789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae); McDowell & Woffenden, (2003) Trends Biotechnol.21(4): 178-83 and Toyoda et al., (2002) Transgenic Res. 11(6):567-82. Aplant resistant to a disease is one that is more resistant to a pathogenas compared to the wild type plant.

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence ofa Bt delta-endotoxin gene. Moreover, DNA molecules encodingdelta-endotoxin genes can be purchased from American Type CultureCollection (Rockville, Md.), for example, under ATCC Accession Nos.40098, 67136, 31995 and 31998. Other examples of Bacillus thuringiensistransgenes being genetically engineered are given in the followingpatents and patent applications and hereby are incorporated by referencefor this purpose: U.S. Pat. Nos. 5,188,960; 5,689,052; 5,880,275; WO91/14778; WO 99/31248; WO 01/12731; WO 99/24581; WO 97/40162 and U.S.application Ser. Nos. 10/032,717; 10/414,637; and 10/606,320.

(C) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344: 458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

(D) An insect-specific peptide which, upon expression, disrupts thephysiology of the affected pest. For example, see the disclosures ofRegan, J. Biol. Chem. 269: 9 (1994) (expression cloning yields DNAcoding for insect diuretic hormone receptor); Pratt et al., Biochem.Biophys. Res. Comm. 163: 1243 (1989) (an allostatin is identified inDiploptera puntata); Chattopadhyay et al. (2004) Critical Reviews inMicrobiology 30 (1): 33-54 2004; Zjawiony (2004) J Nat Prod 67 (2):300-310; Carlini & Grossi-de-Sa (2002) Toxicon, 40 (11): 1515-1539;Ussuf et al. (2001) Curr Sci. 80 (7): 847-853; and Vasconcelos &Oliveira (2004) Toxicon 44 (4): 385-403. See also U.S. Pat. No.5,266,317 to Tomalski et al., who disclose genes encodinginsect-specific toxins.

(E) An enzyme responsible for a hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(F) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTApplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21: 673 (1993), who provide the nucleotide sequenceof the parsley ubi4-2 polyubiquitin gene, U.S. application Ser. Nos.10/389,432, 10/692,367, and U.S. Pat. No. 6,563,020.

(G) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24: 757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

(H) A hydrophobic moment peptide. See PCT Application WO 95/16776 andU.S. Pat. No. 5,580,852 (disclosure of peptide derivatives ofTachyplesin which inhibit fungal plant pathogens) and PCT Application WO95/18855 and U.S. Pat. No. 5,607,914 (teaches synthetic antimicrobialpeptides that confer disease resistance).

(I) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993),of heterologous expression of a cecropin-beta lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

(J) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28: 451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

(K) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium On MolecularPlant-Microbe Interactions (Edinburgh, Scotland, 1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

(L) A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366: 469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(M) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo alpha-1,4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient release bysolubilizing plant cell wall homo-alpha-1,4-D-galacturonase. See Lamb etal., Bio/Technology 10: 1436 (1992). The cloning and characterization ofa gene which encodes a bean endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., Plant J. 2: 367 (1992).

(N) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10: 305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

(O) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, S., Current Biology,5(2):128-131 (1995), Pieterse & Van Loon (2004) Curr. Opin. Plant Bio.7(4):456-64 and Somssich (2003) Cell 113(7):815-6.

(P) Antifungal genes (Cornelissen and Melchers, Pl. Physiol.101:709-712, (1993) and Parijs et al., Planta 183:258-264, (1991) andBushnell et al., Can. J. of Plant Path. 20(2):137-149 (1998). Also seeU.S. application Ser. No. 09/950,933.

(Q) Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see U.S. Pat. No. 5,792,931.

(R) Cystatin and cysteine proteinase inhibitors. See U.S. applicationSer. No. 10/947,979.

(S) Defensin genes. See WO 03/000863 and U.S. application Ser. No.10/178,213.

(T) Genes conferring resistance to nematodes. See WO 03/033651 and Urwinet al., Planta 204:472-479 (1998), Williamson (1999) Curr Opin PlantBio. 2(4):327-31.

(U) Genes such as rcg1 conferring resistance to Anthracnose stalk rot,which is caused by the fungus Colletotrichum graminiola. See M. Jung etal., Generation-means analysis and quantitative trait locus mapping ofAnthracnose Stalk Rot genes in Maize, Theor. Appl. Genet. (1994)89:413-418 which is incorporated by reference for this purpose, as wellas U.S. Patent Application 60/675,664, which is also incorporated byreference for this purpose.

2. Transgenes That Confer Resistance To A Herbicide, For Example:

(A) A herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7: 1241 (1988), and Miki et al., Theor. Appl. Genet. 80: 449(1990), respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659;5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107;5,928,937; and 5,378,824; and international publication WO 96/33270,which are incorporated herein by reference for this purpose.

(B) Glyphosate (resistance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP), and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin acetyl transferase (bar) genes), andpyridinoxy or phenoxy proprionic acids and cycloshexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah et al., which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate resistance. U.S. Pat. No. 5,627,061 to Barryet al. also describes genes encoding EPSPS enzymes. See also U.S. Pat.Nos. 6,566,587; 6,338,961; 6,248,876 B1; 6,040,497; 5,804,425;5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835;5,866,775; 6,225,114 B1; 6,130,366; 5,310,667; 4,535,060; 4,769,061;5,633,448; 5,510,471; Re. 36,449; RE 37,287 E; and 5,491,288; andinternational publications EP1173580; WO 01/66704; EP1173581 andEP1173582, which are incorporated herein by reference for this purpose.Glyphosate resistance is also imparted to plants that express a genethat encodes a glyphosate oxido-reductase enzyme as described more fullyin U.S. Pat. Nos. 5,776,760 and 5,463,175, which are incorporated hereinby reference for this purpose. In addition glyphosate resistance can beimparted to plants by the over expression of genes encoding glyphosateN-acetyltransferase. See, for example, U.S. application Ser. Nos.01/46227; 10/427,692 and 10/427,692. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC Accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European Patent Application No. 0 333 033 to Kumadaet al. and U.S. Pat. No. 4,975,374 to Goodman et al. disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in European PatentNo. 0 242 246 and 0 242 236 to Leemans et al. De Greef et al.,Bio/Technology 7: 61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. See also, U.S. Pat. Nos. 5,969,213;5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477;5,646,024; 6,177,616 B1; and 5,879,903, which are incorporated herein byreference for this purpose. Exemplary genes conferring resistance tophenoxy proprionic acids and cycloshexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83: 435 (1992).

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J. 285:173 (1992).

(D) Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants (see, e.g., Hattori et al. (1995)Mol Gen Genet 246:419). Other genes that confer resistance to herbicidesinclude: a gene encoding a chimeric protein of rat cytochrome P4507A1and yeast NADPH-cytochrome P450 oxidoreductase (Shiota et al. (1994)Plant Physiol. 106(1):17-23), genes for glutathione reductase andsuperoxide dismutase (Aono et al. (1995) Plant Cell Physiol. 36:1687,and genes for various phosphotransferases (Datta et al. (1992) PlantMol. Biol. 20:619).

(E) Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1;and 5,767,373; and international publication WO 01/12825.

3. Transgenes That Confer Or Contribute To An Altered GrainCharacteristic, Such As:

(A) Altered fatty acids, for example, by

-   -   (1) Down-regulation of stearoyl-ACP desaturase to increase        stearic acid content of the plant. See Knultzon et al., Proc.        Natl. Acad. Sci. USA 89: 2624 (1992) and WO99/64579 (Genes for        Desaturases to Alter Lipid Profiles in Corn),    -   (2) Elevating oleic acid via FAD-2 gene modification and/or        decreasing linolenic acid via FAD-3 gene modification (see U.S.        Pat. Nos. 6,063,947; 6,323,392; 6,372,965 and WO 93/11245),    -   (3) Altering conjugated linolenic or linoleic acid content, such        as in WO 01/12800,    -   (4) Altering LEC1, AGP, Dek1, Superal1, mi1ps, various Ipa genes        such as Ipa1, Ipa3, hpt or hggt. For example, see WO 02/42424,        WO 98/22604, WO 03/011015, WO02/057439, WO03/011015, U.S. Pat.        Nos. 6,423,886, 6,197,561, 6,825,397, and U.S. Application        Serial Nos. US2003/0079247, US2003/0204870, and        Rivera-Madrid, R. et al. Proc. Natl. Acad. Sci. 92:5620-5624        (1995).

(B) Altered phosphorus content, for example, by the

-   -   (1) Introduction of a phytase-encoding gene would enhance        breakdown of phytate, adding more free phosphate to the        transformed plant. For example, see Van Hartingsveldt et al.,        Gene 127: 87 (1993), for a disclosure of the nucleotide sequence        of an Aspergillus niger phytase gene.    -   (2) Up-regulation of a gene that reduces phytate content. In        maize, this, for example, could be accomplished, by cloning and        then re-introducing DNA associated with one or more of the        alleles, such as the LPA alleles, identified in maize mutants        characterized by low levels of phytic acid, such as in Raboy et        al., Maydica 35: 383 (1990) and/or by altering inositol kinase        activity as in WO 02/059324, US2003/0009011, WO 03/027243,        US2003/0079247, WO 99/05298, US6197561, US6291224, US6391348,        WO2002/059324, US2003/0079247, WO98/45448, WO99/55882,        WO01/04147.

(C) Altered carbohydrates effected, for example, by altering a gene foran enzyme that affects the branching pattern of starch, a gene alteringthioredoxin such as NTR and/or TRX (see U.S. Pat. No. 6,531,648, whichis incorporated by reference for this purpose) and/or a gamma zein knockout or mutant such as cs27 or TUSC27 or en27 (See U.S. Pat. No.6,858,778 and US2005/0160488, US2005/0204418, which are incorporated byreference for this purpose). See Shiroza et al., J. Bacteriol. 170: 810(1988) (nucleotide sequence of Streptococcus mutans fructosyltransferasegene), Steinmetz et al., Mol. Gen. Genet. 200: 220 (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al.,Bio/Technology 10: 292 (1992) (production of transgenic plants thatexpress Bacillus licheniformis alpha-amylase), Elliot et al., PlantMolec. Biol. 21: 515 (1993) (nucleotide sequences of tomato invertasegenes), Søgaard et al., J. Biol. Chem. 268: 22480 (1993) (site-directedmutagenesis of barley alpha-amylase gene), and Fisher et al., PlantPhysiol. 102: 1045 (1993) (maize endosperm starch branching enzyme II),WO 99/10498 (improved digestibility and/or starch extraction throughmodification of UDP-D-xylose 4-epimerase, Fragile 1 and 2, Ref1, HCHL,C4H), U.S. Pat. No. 6,232,529 (method of producing high oil seed bymodification of starch levels (AGP)). The fatty acid modification genesmentioned above may also be used to affect starch content and/orcomposition through the interrelationship of the starch and oilpathways.

(D) Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, see U.S. Pat. No. 6,787,683,US2004/0034886 and WO 00/68393 involving the manipulation of antioxidantlevels through alteration of a phytl prenyl transferase (ppt), WO03/082899 through alteration of a homogentisate geranyl geranyltransferase (hggt).

(E) Altered essential seed amino acids. For example, see U.S. Pat. No.6,127,600 (method of increasing accumulation of essential amino acids inseeds), U.S. Pat. No. 6,080,913 (binary methods of increasingaccumulation of essential amino acids in seeds), U.S. Pat. No. 5,990,389(high lysine), WO99/40209 (alteration of amino acid compositions inseeds), WO99/29882 (methods for altering amino acid content ofproteins), U.S. Pat. No. 5,850,016 (alteration of amino acidcompositions in seeds), WO98/20133 (proteins with enhanced levels ofessential amino acids), U.S. Pat. No. 5,885,802 (high methionine), U.S.Pat. No. 5,885,801 (high threonine), U.S. Pat. No. 6,664,445 (plantamino acid biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increasedlysine and threonine), U.S. Pat. No. 6,441,274 (plant tryptophansynthase beta subunit), U.S. Pat. No. 6,346,403 (methionine metabolicenzymes), U.S. Pat. No. 5,939,599 (high sulfur), U.S. Pat. No. 5,912,414(increased methionine), WO98/56935 (plant amino acid biosyntheticenzymes), WO98/45458 (engineered seed protein having higher percentageof essential amino acids), WO98/42831 (increased lysine), U.S. Pat. No.5,633,436 (increasing sulfur amino acid content), U.S. Pat. No.5,559,223 (synthetic storage proteins with defined structure containingprogrammable levels of essential amino acids for improvement of thenutritional value of plants), WO96/01905 (increased threonine),WO95/15392 (increased lysine), US2003/0163838, US2003/0150014,US2004/0068767, U.S. Pat. No. 6,803,498, WO01/79516, and WO00/09706 (CesA: cellulose synthase), U.S. Pat. No. 6,194,638 (hemicellulose), U.S.Pat. No. 6,399,859 and US2004/0025203 (UDPGdH), U.S. Pat. No. 6,194,638(RGP).

4. Genes that Control Male-sterility:

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

(A) Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT (WO 01/29237).

(B) Introduction of various stamen-specific promoters (WO 92/13956, WO92/13957).

(C) Introduction of the barnase and the barstar gene (Paul et al. PlantMol. Biol. 19:611-622, 1992).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341; 6,297,426; 5,478,369;5,824,524; 5,850,014; and 6,265,640; all of which are herebyincorporated by reference.

5. Genes that create a site for site specific DNA integration. Thisincludes the introduction of FRT sites that may be used in the FLP/FRTsystem and/or Lox sites that may be used in the Cre/Loxp system. Forexample, see Lyznik, et al., Site-Specific Recombination for GeneticEngineering in Plants, Plant Cell Rep (2003) 21:925-932 and WO 99/25821which are hereby incorporated by reference. Other systems that may beused include the Gin recombinase of phage Mu (Maeser et al., 1991; VickiChandler, The Maize Handbook ch. 118 (Springer-Verlag 1994), the Pinrecombinase of E. coli (Enomoto et al., 1983), and the R/RS system ofthe pSR1 plasmid (Araki et al., 1992).

6. Genes that affect abiotic stress resistance (including but notlimited to flowering, ear and seed development, enhancement of nitrogenutilization efficiency, altered nitrogen responsiveness, droughtresistance or tolerance, cold resistance or tolerance, and saltresistance or tolerance) and increased yield under stress. For example,see: WO 00/73475 where water use efficiency is altered throughalteration of malate; U.S. Pat. Nos. 5,892,009, 5,965,705, 5,929,305,5,891,859, 6,417,428, 6,664,446, 6,706,866, 6,717,034, WO2000060089,WO2001026459, WO2001035725, WO2001034726, WO2001035727, WO2001036444,WO2001036597, WO2001036598, WO2002015675, WO2002017430, WO2002077185,WO2002079403, WO2003013227, WO2003013228, WO2003014327, WO2004031349,WO2004076638, WO9809521, and WO9938977 describing genes, including CBFgenes and transcription factors effective in mitigating the negativeeffects of freezing, high salinity, and drought on plants, as well asconferring other positive effects on plant phenotype; US2004/0148654 andWO01/36596 where abscisic acid is altered in plants resulting inimproved plant phenotype such as increased yield and/or increasedtolerance to abiotic stress; WO2000/006341, WO04/090143, U.S.application Ser. Nos. 10/817,483 and 09/545,334 where cytokininexpression is modified resulting in plants with increased stresstolerance, such as drought tolerance, and/or increased yield. Also seeWO0202776, WO2003052063, JP2002281975, U.S. Pat. No. 6,084,153,WO0164898, U.S. Pat. Nos. 6,177,275, and 6,107,547 (enhancement ofnitrogen utilization and altered nitrogen responsiveness). For ethylenealteration, see US20040128719, US20030166197 and WO200032761. For planttranscription factors or transcriptional regulators of abiotic stress,see e.g. US20040098764 or US20040078852.

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants, see e.g.WO97/49811 (LHY), WO98/56918 (ESD4), WO97/10339 and U.S. Pat. No.6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO96/14414 (CON),WO96/38560, WO01/21822 (VRN1), WO00/44918 (VRN2), WO99/49064 (GI),WO00/46358 (FRI), WO97/29123, U.S. Pat. Nos. 6,794,560, 6,307,126 (GAI),WO99/09174 (D8 and Rht), and WO2004076638 and WO2004031349(transcription factors).

Genetic Marker Profile Through SSR

The present invention comprises a hybrid corn plant which ischaracterized by the molecular and physiological data presented hereinand in the representative sample of said hybrid and of the inbredparents of said hybrid deposited with the ATCC.

To select and develop a superior hybrid, it is necessary to identify andselect genetically unique individuals that occur in a segregatingpopulation. The segregating population is the result of a combination ofcrossover events plus the independent assortment of specificcombinations of alleles at many gene loci that results in specific andunique genotypes. Once such a line is developed its value to society issubstantial since it is important to advance the germplasm base as awhole in order to maintain or improve traits such as yield, diseaseresistance, pest resistance and plant performance in extreme weatherconditions. Backcross trait conversions are routinely used to add ormodify one or a few traits of such a line and this further enhances itsvalue and usefulness to society. The genetic variation among individualprogeny of a breeding cross allows for the identification of rare andvaluable new genotypes. Once identified, it is possible to utilizeroutine and predictable breeding methods to develop progeny that retainthe rare and valuable new genotypes developed by the initial breeder.

Phenotypic traits exhibited by 32H20 can be used to characterize thegenetic contribution of 32H20 to progeny lines developed through the useof 32H20. Quantitative traits including, but not limited to, yield,maturity, stay green, root lodging, stalk lodging, and early growth aretypically governed by multiple genes at multiple loci. A breeder willcommonly work to combine a specific trait of an undeveloped variety ofthe species, such as a high level of resistance to a particular disease,with one or more of the elite agronomic characteristics (yield,maturity, plant size, lodging resistance, etc.) needed for use as acommercial variety. This combination, once developed, provides avaluable source of new germplasm for further breeding. For example, itmay take 10-15 years and significant effort to produce such acombination, yet progeny may be developed that retain this combinationin as little as 2-5 years and with much less effort. 32H20 progenyplants that retain the same degree of phenotypic expression of thesequantitative traits as 32H20 have received significant genotypic andphenotypic contribution from 32H20. This characterization is enhancedwhen such quantitative trait is not exhibited in non-32H20 breedingmaterial used to develop the 32H20 progeny.

As discussed, supra, in addition to phenotypic observations, a plant canalso be described by its genotype. The genotype of a plant can bedescribed through a genetic marker profile which can identify plants ofthe same variety, a related variety or be used to determine or validatea pedigree. Genetic marker profiles can be obtained by techniques suchas Restriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), Simple Sequence Repeats (SSRs) which are also referred to asMicrosatellites, and Single Nucleotide Polymorphisms (SNPs). Forexample, see Berry, Don, et al., “Assessing Probability of AncestryUsing Simple Sequence Repeat Profiles: Applications to Maize Hybrids andInbreds”, Genetics, 2002, 161: 813-824, and Berry, Don et al.,“Assessing Probability of Ancestry Using Simple Sequence RepeatProfiles: Applications to Maize Inbred Lines and Soybean Varieties”,Genetics, 2003, 165: 331-342, which are incorporated by reference hereinin their entirety.

Particular markers used for these purposes are not limited to the set ofmarkers disclosed herein, but may include any type of marker and markerprofile which provides a means of distinguishing varieties. In additionto being used for identification of inbred parents, hybrid variety32H20, a hybrid produced through the use of 32H20 or its parents, andthe identification or verification of pedigree for progeny plantsproduced through the use of 32H20, the genetic marker profile is alsouseful in breeding and developing an introgressed trait conversion of32H20.

Means of performing genetic marker profiles using SSR polymorphisms arewell known in the art. SSRs are genetic markers based on polymorphismsin repeated nucleotide sequences, such as microsatellites. A markersystem based on SSRs can be highly informative in linkage analysisrelative to other marker systems in that multiple alleles may bepresent. Another advantage of this type of marker is that, through useof flanking primers, detection of SSRs can be achieved, for example, bythe polymerase chain reaction (PCR), thereby eliminating the need forlabor-intensive Southern hybridization. The PCR™ detection is done byuse of two oligonucleotide primers flanking the polymorphic segment ofrepetitive DNA. Repeated cycles of heat denaturation of the DNA followedby annealing of the primers to their complementary sequences at lowtemperatures, and extension of the annealed primers with DNA polymerase,comprise the major part of the methodology.

Following amplification, markers can be scored by gel electrophoresis ofthe amplification products. Scoring of marker genotype is based on thesize of the amplified fragment, which may be measured by the base pairweight or molecular weight of the fragment. While variation in theprimer used or in laboratory procedures can affect the reportedmolecular weight, relative values should remain constant regardless ofthe specific primer or laboratory used. When comparing lines it ispreferable if all SSR profiles are performed in the same lab. An SSRservice is available to the public on a contractual basis by DNALandmarks in Saint-Jean-sur-Richelieu, Quebec, Canada.

Primers used for the SSRs suggested herein are publicly available andmay be found in the Maize GDB on the World Wide Web at maizegdb.org(sponsored by the USDA Agricultural Research Service), in Sharopova etal. (Plant Mol. Biol. 48(5-6):463-481), Lee et al. (Plant Mol. Biol.48(5-6); 453-461). Primers may be constructed from publicly availablesequence information. Some marker information may be available from DNALandmarks.

A genetic marker profile of a hybrid should be the sum of its inbredparents, e.g., if one inbred parent is homozygous for allele x at aparticular locus, and the other inbred parent is homozygous for allele yat that locus, the F1 hybrid will be x.y (heterozygous) at that locus.The profile can therefore be used to identify the inbred parents ofhybrid 32H20. The determination of the male set of alleles and thefemale set of alleles may be made by profiling the hybrid and thepericarp of the hybrid seed, which is composed of maternal parent cells.The paternal parent profile is obtained by subtracting the pericarpprofile from the hybrid profile.

In addition, plants and plant parts substantially benefiting from theuse of 32H20 in their development, such as 32H20 comprising anintrogressed trait through backcross conversion or transformation, maybe identified by having an SSR molecular marker profile with a highpercent identity to 32H20, such as at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 99.5% identity to 32H20.

The SSR profile of 32H20 also can be used to identify essentiallyderived varieties and other progeny lines developed from the use of32H20, as well as cells and other plant parts thereof. Progeny plantsand plant parts produced using 32H20 may be identified by having amolecular marker profile of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%genetic contribution from hybrid maize plant 32H20, as measured byeither percent identity or percent similarity.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. 32H20 is suitable for use in a recurrentselection program. The method entails individual plants crosspollinating with each other to form progeny. The progeny are grown andthe superior progeny selected by any number of selection methods, whichinclude individual plant, half-sib progeny, full-sib progeny, selfedprogeny and topcrossing. The selected progeny are cross pollinated witheach other to form progeny for another population. This population isplanted and again superior plants are selected to cross pollinate witheach other. Recurrent selection is a cyclical process and therefore canbe repeated as many times as desired. The objective of recurrentselection is to improve the traits of a population. The improvedpopulation can then be used as a source of breeding material to obtaininbred lines to be used in hybrids or used as parents for a syntheticcultivar. A synthetic cultivar is the resultant progeny formed by theintercrossing of several selected inbreds.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection seeds fromindividuals are selected based on phenotype and/or genotype. Theseselected seeds are then bulked and used to grow the next generation.Bulk selection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Instead of self pollination, directed pollination could beused as part of the breeding program.

Mutation Breeding

Mutation breeding is one of many methods that could be used to introducenew traits into 32H20. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g.cobalt 60 or cesium 137), neutrons, (product of nuclear fission byuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (preferably from 2500 to 2900 nm), or chemical mutagens (suchas base analogues (5-bromo-uracil), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethylenamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques, such as backcrossing. Details of mutation breedingcan be found in “Principles of Cultivar Development” Fehr, 1993Macmillan Publishing Company, the disclosure of which is incorporatedherein by reference. In addition, mutations created in other lines maybe used to produce a backcross conversion of 32H20 that comprises suchmutation.

INDUSTRIAL APPLICABILITY

Maize is used as human food, livestock feed, and as raw material inindustry. The food uses of maize, in addition to human consumption ofmaize kernels, include both products of dry- and wet-milling industries.The principal products of maize dry milling are grits, meal and flour.The maize wet-milling industry can provide maize starch, maize syrups,and dextrose for food use. Maize oil is recovered from maize germ, whichis a by-product of both dry- and wet-milling industries.

Maize, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs, and poultry.

Industrial uses of maize include production of ethanol, maize starch inthe wet-milling industry and maize flour in the dry-milling industry.The industrial applications of maize starch and flour are based onfunctional properties, such as viscosity, film formation, adhesiveproperties, and ability to suspend particles. The maize starch and flourhave application in the paper and textile industries. Other industrialuses include applications in adhesives, building materials, foundrybinders, laundry starches, explosives, oil-well muds, and other miningapplications.

Plant parts other than the grain of maize are also used in industry: forexample, stalks and husks are made into paper and wallboard and cobs areused for fuel and to make charcoal.

The seed of the hybrid maize plant, the plant produced from the seed, aplant produced from crossing of maize hybrid plant 32H20 and variousparts of the hybrid maize plant and transgenic versions of theforegoing, can be utilized for human food, livestock feed, and as a rawmaterial in industry.

REFERENCES

-   Aukerman, M. J. et al. (2003) “Regulation of Flowering Time and    Floral Organ Identity by a MicroRNA and Its APETALA2-like Target    Genes” The Plant Cell 15:2730-2741-   Berry et al., “Assessing Probability of Ancestry Using Simple    Sequence Repeat Profiles: Applications to Maize Hybrids and    Inbreds”, Genetics 161:813-824 (2002)-   Berry et al., “Assessing Probability of Ancestry Using Simple    Sequence Repeat Profiles: Applications to Maize Inbred Lines and    Soybean Varieties” Genetics 165:331-342 (2003)-   Boppenmaier, et al., “Comparisons Among Strains of Inbreds for    RFLPs”, Maize Genetics Cooperative Newsletter, 65:1991, p. 90-   Conger, B. V., et al. (1987) “Somatic Embryogenesis From Cultured    Leaf Segments of Zea Mays”, Plant Cell Reports, 6:345-347-   Duncan, D. R., et al. (1985) “The Production of Callus Capable of    Plant Regeneration From Immature Embryos of Numerous Zea Mays    Genotypes”, Planta, 165:322-332-   Edallo, et al. (1981) “Chromosomal Variation and Frequency of    Spontaneous Mutation Associated with in Vitro Culture and Plant    Regeneration in Maize”, Maydica, XXVI: 39-56-   Fehr, Walt, Principles of Cultivar Development, pages 261-286 (1987)-   Green, et al. (1975) “Plant Regeneration From Tissue Cultures of    Maize”, Crop Science, Vol. 15, pages 417-421-   Green, C. E., et al. (1982) “Plant Regeneration in Tissue Cultures    of Maize” Maize for Biological Research, pages 367-372-   Hallauer, A. R. et al. (1988) “Corn Breeding” Corn and Corn    Improvement, No. 18, pages 463-481-   Lee, Michael (1994) “Inbred Lines of Maize and Their Molecular    Markers”, The Maize Handbook, Ch. 65:423-432-   Meghji, M. R., et al. (1984) “Inbreeding Depression, Inbred & Hybrid    Grain Yields, and Other Traits of Maize Genotypes Representing Three    Eras”, Crop Science, Vol. 24, pages 545-549-   Openshaw, S. J., et al. (1994) “Marker-assisted selection in    backcross breeding”, pages 41-43. In Proceedings of the Symposium    Analysis of Molecular Marker Data. 5-7 Aug. 1994. Corvallis, Oreg.,    American Society for Horticultural Science/Crop Science Society of    America-   Phillips, et al. (1988) “Cell/Tissue Culture and In Vitro    Manipulation”, Corn & Corn Improvement, 3rd Ed., ASA Publication,    No. 18, pages 345-387-   Poehlman et al (1995) Breeding Field Crop, 4th Ed., Iowa State    University Press, Ames, Iowa, pages 132-155 and 321-344-   Rao, K. V., et al., (1986) “Somatic Embryogenesis in Glume Callus    Cultures”, Maize Genetics Cooperative Newsletter, No. 60, pages    64-65-   Sass, John F. (1977) “Morphology”, Corn & Corn Improvement, ASA    Publication, Madison, Wis. pages 89-109-   Smith, J. S. C., et al., “The Identification of Female Selfs in    Hybrid Maize: A Comparison Using Electrophoresis and Morphology”,    Seed Science and Technology 14, 1-8-   Songstad, D. D. et al. (1988) “Effect of    ACC(1-aminocyclopropane-1-carboyclic acid), Silver Nitrate &    Norbonadiene on Plant Regeneration From Maize Callus Cultures”,    Plant Cell Reports, 7:262-265-   Tomes, et al. (1985) “The Effect of Parental Genotype on Initiation    of Embryogenic Callus From Elite Maize (Zea Mays L.) Germplasm”,    Theor. Appl. Genet., Vol. 70, p. 505-509-   Troyer, et al. (1985) “Selection for Early Flowering in Corn: 10    Late Synthetics”, Crop Science, Vol. 25, pages 695-697-   Umbeck, et al. (1983) “Reversion of Male-Sterile T-Cytoplasm Maize    to Male Fertility in Tissue Culture”, Crop Science, Vol. 23, pages    584-588-   Wan et al., “Efficient Production of Doubled Haploid Plants Through    Colchicine Treatment of Anther-Derived Maize Callus”, Theoretical    and Applied Genetics, 77:889-892, 1989-   Wright, Harold (1980) “Commercial Hybrid Seed Production”,    Hybridization of Crop Plants, Ch. 8:161-176-   Wych, Robert D. (1988) “Production of Hybrid Seed”, Corn and Corn    Improvement, Ch. 9, pages 565-607    Deposits

Applicant(s) have made a deposit of at least 2500 seeds of hybrid maize32H20 with the American Type Culture Collection (ATCC), Manassas, Va.20110 USA, ATCC Deposit No. ______. The seeds deposited with the ATCC on______ were taken from the deposit maintained by Pioneer Hi-BredInternational, Inc., 7250 NW 62^(nd) Avenue, Johnston, Iowa 50131-1000since prior to the filing date of this application. Access to thisdeposit will be available during the pendency of the application to theCommissioner of Patents and Trademarks and persons determined by theCommissioner to be entitled thereto upon request. Upon allowance of anyclaims in the application, the Applicant(s) will make available to thepublic, pursuant to 37 C.F.R. § 1.808, sample(s) of the deposit of atleast 2500 seeds of hybrid maize 32H20 with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209.This deposit of seed of hybrid maize 32H20 will be maintained in theATCC depository, which is a public depository, for a period of 30 years,or 5 years after the most recent request, or for the enforceable life ofthe patent, whichever is longer, and will be replaced if it becomesnonviable during that period. Additionally, Applicant(s) have satisfiedall the requirements of 37 C.F.R. §§1.801-1.809, including providing anindication of the viability of the sample upon deposit. Applicant(s)have no authority to waive any restrictions imposed by law on thetransfer of biological material or its transportation in commerce.Applicant(s) do not waive any infringement of their rights granted underthis patent or rights applicable to Hybrid Maize 32H20 under the PlantVariety Protection Act (7 USC 2321 et seq.). TABLE 1 VARIETY DESCRIPTIONINFORMATION 32H20 AVG STDEV N 1. TYPE: (Describe intermediate types incomments section) 1 = Sweet, 2 = Dent, 3 = Flint, 4 = Flour, 5 = Pop and3 6 = Ornamental. Comments: Flint-Dent 2. MATURITY: DAYS HEAT UNITS DaysH. Units Emergence to 50% of plants in silk 63 1,403 Emergence to 50% ofplants in pollen shed 63 1,395 10% to 90% pollen shed 2 56 50% Silk toharvest at 25% moisture 3. PLANT: Plant Height (to tassel tip) (cm)306.3 31.39 15 Ear Height (to base of top ear node) (cm) 118.0 22.67 15Length of Top Ear Internode (cm) 21.3 1.75 15 Average Number of Tillersper Plant 0.0 0.02 3 Average Number of Ears per Stalk 1.0 0.03 3Anthocyanin of Brace Roots: 1 = Absent, 2 = Faint, 2 3 = Moderate, 4 =Dark 4. LEAF: Width of Ear Node Leaf (cm) 10.5 1.06 15 Length of EarNode Leaf (cm) 96.6 4.19 15 Number of Leaves above Top Ear 7.3 0.59 15Leaf Angle: (at anthesis, 2nd leaf above ear to 28.1 4.41 15 stalk aboveleaf) (Degrees) *Leaf Color: V. Dark Green Munsell: 7.5GY36 Leaf SheathPubescence: 1 = none to 9 = like peach fuzz 5 5. TASSEL: Number ofPrimary Lateral Branches 7.2 1.52 15 Branch Angle from Central Spike52.2 17.86 15 Tassel Length: (from peduncle node to tassel tip), (cm).68.9 4.73 15 Pollen Shed: 0 = male sterile, 9 = heavy shed 6 *AntherColor: Pale Yellow Munsell: 10YR86 *Glume Color: Med. Green Munsell:5GY66 *Bar Glumes (glume bands): 1 = absent, 2 = present 1 PeduncleLength: (from top leaf node to lower florets or 26.1 2.37 15 branches),(cm). 6a. EAR (Unhusked ear) *Silk color: Light Green Munsell: 5GY76 (3days after silk emergence) *Fresh husk color: Med. Green Munsell: 5GY68*Dry husk color: Buff Munsell: 10YR84 (65 days after 50% silking) Earposition at dry husk stage: 1 = upright, 2 = horizontal, 1 3 = pendantHusk Tightness: (1 = very loose, 9 = very tight) 4 Husk Extension (atharvest): 1 = short(ears exposed), 1 2 = medium (<8 cm), 3 = long (8-10cm), 4 = v. long (>10 cm) 6b. EAR (Husked ear data) Ear Length (cm):19.9 1.03 15 Ear Diameter at mid-point (mm) 49.7 2.06 15 Ear Weight(gm): 259.1 28.31 15 Number of Kernel Rows: 15.2 1.82 15 Kernel Rows: 1= indistinct, 2 = distinct 2 Row Alignment: 1 = straight, 2 = slightlycurved, 3 = spiral 2 Shank Length (cm): 10.4 1.88 15 Ear Taper: 1 =slight cylind., 2 = average, 3 = extreme conic. 2 7. KERNEL (Dried):Kernel Length (mm): 13.4 0.63 15 Kernel Width (mm): 8.7 0.72 15 KernelThickness (mm): 4.2 0.41 15 Round Kernels (shape grade) (%) 13.6 4.99 3Aleurone Color Pattern: 1 = homozygous, 2 = segregating 1 *AleuroneColor: Yellow Munsell: 10YR814 *Hard Endo. Color: Yellow Munsell:10YR714 Endosperm Type: 3 1 = sweet (su1), 2 = extra sweet (sh2), 3 =normal starch, 4 = high amylose starch, 5 = waxy starch, 6 = highprotein, 7 = high lysine, 8 = super sweet (se), 9 = high oil, 10 = otherWeight per 100 Kernels (unsized sample) (gm): 36.3 1.15 3 8. COB: *CobDiameter at mid-point (mm): 25.7 1.40 15 *Cob Color: Pink OrangeMunsell: 2.5YR48 10. DISEASE RESISTANCE: (Rate from 1 = most-susceptableto 9 = most-resistant. Leave blank if not tested, leave race or strainoptions blank if polygenic.) A. LEAF BLIGHTS, WILTS, AND LOCAL INFECTIONDISEASES Anthracnose Leaf Blight (Colletotrichum graminicola) CommonRust (Puccinia sorghi) Common Smut (Ustilago maydis) Eyespot (Kabatiellazeae) Goss's Wilt (Clavibacter michiganense spp. 6 Gray Leaf Spot(Cercospora zeae-maydis) Helminthosporium Leaf Spot (Bipolaris zeicola)Race: 4 Northern Leaf Blight (Exserohilum turcicum) Race: Southern LeafBlight (Bipolaris maydis) Race: Southern Rust (Puccinia polysora)Stewart's Wilt (Erwinia stewartii) Other (Specify):            B.SYSTEMIC DISEASES Corn Lethal Necrosis (MCMV and MDMV) 26 Head Smut(Sphacelotheca reiliana) (% infected) Maize Chlorotic Dwarf Virus (MDV)Maize Chlorotic Mottle Virus (MCMV) Maize Dwarf Mosaic Virus (MDMV)Sorghum Downy Mildew of Corn (Peronosclerospora sorghi) Other (Specify):           C. STALK ROTS 4 Anthracnose Stalk Rot (Colletotrichumgraminicola) Diplodia Stalk Rot (Stenocarpella maydis) Fusarium StalkRot (Fusarium moniliforme) Gibberella Stalk Rot (Gibberella zeae) Other(Specify):            D. EAR AND KERNEL ROTS Aspergillus Ear and KernelRot (Aspergillus flavus) 4 Diplodia Ear Rot (Stenocarpella maydis)Fusarium Ear and Kernel Rot (Fusarium moniliforme) Gibberella Ear Rot(Gibberella zeae) Other (Specify):            11. INSECT RESISTANCE:(Rate from 1 = most-suscept. to 9 = most-resist., leave blank if nottested.) Corn Worm (Helicoverpa zea)     Leaf Feeding     Silk Feeding    Ear Damage Corn Leaf Aphid (Rophalosiphum maydis) Corn Sap Beetle(Capophilus dimidiatus) European Corn Borer (Ostrinia nubilalis) 1st.Generation (Typically whorl leaf feeding) 2nd. Generation (Typicallyleaf sheath-collar feeding)     Stalk Tunneling     cm tunneled/plantFall armyworm (Spodoptera fruqiperda)     Leaf Feeding     Silk Feeding    mg larval wt. Maize Weevil (Sitophilus zeamaize) Northern Rootworm(Diabrotica barberi) Southern Rootworm (Diabrotica undecimpunctata)Southwestern Corn Borer (Diatreaea grandiosella)     Leaf Feeding    Stalk Tunneling     cm tunneled/plant Two-spotted Spider Mite(Tetranychus utricae) Western Rootworm (Diabrotica virgifrea virgifrea)Other (Specify):            12. AGRONOMIC TRAITS: 6 Staygreen (at 65days after anthesis; rate from 1-worst to 9-excellent) % Dropped Ears(at 65 days after anthesis) % Pre-anthesis Brittle Snapping 3 %Pre-anthesis Root Lodging 8 % Post-anthesis Root Lodging (at 65 daysafter anthesis) 13 % Post-anthesis Stalk Lodging 12,601.0 Kg/ha (Yieldat 12-13% grain moisture)*Munsell Glossy Book of color, (A standard color reference). KollmorgenInst. Corp. New Windsor, NY.

TABLE 2A HYBRID COMPARISON Variety #1: 32H20 Variety #2: 33Y45 YIELDYIELD ESTCNT GDUSHD BU/A 56# BU/A 56# MST EGRWTH COUNT GDU Stat ABS % MNPCT % MN SCORE % MN % MN % MN Mean1 202.0 104.1 104.7 90.4 96.4 102.2Mean2 203.1 104.0 103.5 102.7 96.4 103.9 Locs 118 118 118 15 4 29 Reps132 132 133 16 5 38 Diff −1.1 0.1 −1.2 −12.3 −0.1 −1.7 Prob 0.580 0.9490.198 0.017 0.986 0.000 GDUSLK PLTHT EARHT STKLDG GDU STKCNT CM CMSTAGRN % NOT Stat % MN COUNT % MN % MN % MN SCORE % MN % MN Mean1 102.099.5 100.4 99.8 124.0 112.6 Mean2 103.9 100.4 104.5 107.5 122.3 102.4Locs 28 199 44 41 32 2 Reps 37 292 53 47 34 4 Diff −1.9 −0.9 −4.1 −7.71.7 10.2 Prob 0.000 0.016 0.000 0.000 0.789 0.295 STLLPN TSTWT GLFSPTNLFBLT % NOT LB/BU SCORE SCORE SLFBLT GOSWLT Stat % MN ABS ABS ABS SCOREABS SCORE ABS Mean1 106.4 56.9 5.8 3.9 5.3 7.5 Mean2 119.5 56.6 6.1 4.75.3 7.0 Locs 17 81 9 12 3 3 Reps 31 91 14 18 4 6 Diff −13.1 0.3 −0.3−0.8 0.0 0.5 Prob 0.081 0.096 0.508 0.014 1.000 0.478 ANTROT FUSERSDIPERS COMRST HSKCVR SCORE SCORE SCORE SCORE SCORE GIBROT Stat ABS ABSABS ABS ABS SCORE ABS Mean1 4.4 5.2 3.7 2.5 4.9 5.0 Mean2 4.9 4.8 5.13.0 4.6 8.0 Locs 6 3 9 1 14 2 Reps 11 6 18 2 14 4 Diff −0.5 0.3 −1.3−0.5 0.4 −3.0 Prob 0.275 0.423 0.004 — 0.239 0.105 BRTSTK HDSMT ERTLPNLRTLPN DIPROT % NOT % NOT % NOT % NOT STLPCN Stat SCORE ABS ABS ABS ABSABS % NOT ABS Mean1 4.0 94.8 74.6 96.5 92.1 85.9 Mean2 4.5 97.3 88.293.1 90.2 85.9 Locs 1 13 8 9 28 28 Reps 2 19 22 11 34 34 Diff −0.5 −2.5−13.6 3.4 2.0 0.0 Prob — 0.166 0.079 0.231 0.588 1.000

TABLE 2B HYBRID COMPARISON Variety #1: 32H20 Variety #2: 32K22 YIELDYIELD ESTCNT GDUSHD BU/A 56# BU/A 56# MST EGRWTH COUNT GDU Stat ABS % MNPCT % MN SCORE % MN % MN % MN Mean1 202.3 104.1 104.7 90.4 96.4 101.2Mean2 201.7 103.4 104.8 109.4 94.8 99.9 Locs 116 116 116 15 4 25 Reps131 131 131 16 5 34 Diff 0.7 0.7 0.1 −18.9 1.5 1.3 Prob 0.757 0.5300.899 0.003 0.653 0.003 GDUSLK PLTHT EARHT STKLDG GDU STKCNT CM CMSTAGRN % NOT Stat % MN COUNT % MN % MN % MN SCORE % MN % MN Mean1 100.999.3 100.4 99.8 123.9 112.6 Mean2 99.9 100.0 101.3 104.5 102.5 99.8 Locs24 193 44 41 31 2 Reps 33 284 52 46 33 4 Diff 1.0 −0.6 −0.9 −4.7 21.412.8 Prob 0.040 0.116 0.157 0.000 0.002 0.295 STLLPN TSTWT GLFSPT NLFBLT% NOT LB/BU SCORE SCORE SLFBLT GOSWLT Stat % MN ABS ABS ABS SCORE ABSSCORE ABS Mean1 106.4 56.8 5.8 3.9 5.3 7.5 Mean2 110.0 56.7 4.8 4.2 4.86.7 Locs 17 77 9 12 3 3 Reps 31 88 15 18 4 6 Diff −3.6 0.1 1.0 −0.3 0.50.8 Prob 0.675 0.419 0.007 0.256 0.423 0.199 ANTROT FUSERS DIPERS COMRSTHSKCVR SCORE SCORE SCORE SCORE SCORE GIBROT Stat ABS ABS ABS ABS ABSSCORE ABS Mean1 4.4 5.2 3.7 2.5 4.9 5.0 Mean2 4.1 5.7 4.9 3.0 5.7 6.5Locs 6 3 9 1 14 2 Reps 11 6 17 2 14 4 Diff 0.3 −0.5 −1.2 −0.5 −0.8 −1.5Prob 0.175 0.423 0.019 — 0.003 0.205 BRTSTK HDSMT ERTLPN LRTLPN DIPROT %NOT % NOT % NOT % NOT STLPCN Stat SCORE ABS ABS ABS ABS ABS % NOT ABSMean1 4.0 94.8 74.6 96.5 92.1 86.5 Mean2 1.5 97.2 76.5 83.9 82.9 70.9Locs 1 13 8 9 28 27 Reps 2 19 21 11 34 33 Diff 2.5 −2.4 −1.9 12.6 9.315.6 Prob — 0.314 0.752 0.174 0.033 0.004

All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All such publications, patents and patentapplications are incorporated by reference herein for the purpose citedto the same extent as if each was specifically and individuallyindicated to be incorporated by reference herein.

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding. Asis readily apparent to one skilled in the art, the foregoing are onlysome of the methods and compositions that illustrate the embodiments ofthe foregoing invention. It will be apparent to those of ordinary skillin the art that variations, changes, modifications and alterations maybe applied to the compositions and/or methods described herein withoutdeparting from the true spirit, concept and scope of the invention.

1. A seed of hybrid maize variety designated 32H20, representative seedof said variety having been deposited under ATCC Accession No: PTA-XXX.2. A maize plant, or a part thereof, produced by growing the seed ofclaim
 1. 3. Pollen of the plant of claim
 2. 4. An ovule or ovules of theplant of claim
 2. 5. A maize plant, or a part thereof, having all thephysiological and morphological characteristics of the hybrid maizevariety 32H20, representative seed of said variety having been depositedunder ATCC Accession No: PTA-XXXX.
 6. A tissue culture of regenerablecells produced from the plant of claim
 2. 7. Protoplasts or callusproduced from the tissue culture of claim
 6. 8. The tissue culture ofclaim 6, wherein the regenerable cells of the tissue culture areproduced from protoplasts or from tissue of a plant part selected fromthe group consisting of leaf, pollen, embryo, immature embryo,meristematic cells, immature tassels, microspores, root, root tip,anther, silk, flower, kernel, ear, cob, husk and stalk.
 9. A maize plantregenerated from the tissue culture of claim 6, said plant having allthe morphological and physiological characteristics of hybrid maizevariety 32H20, representative seed of said variety having been depositedunder ATCC Accession No: PTA-XXXX.
 10. A process for producing an F1hybrid maize seed, said process comprising crossing the plant of claim 2with a different maize plant and harvesting the F1 hybrid maize seed.11. The process of claim 10, further comprising growing the F1 hybridmaize seed to produce a hybrid maize plant.
 12. A process for producinga maize seed, comprising crossing the plant of claim 2 with itself or adifferent maize plant and harvesting the resultant maize seed.
 13. Theprocess of claim 12, wherein said different maize plant is a haploidinducer.
 14. A process of introducing a desired trait into a hybridmaize variety 32H20 comprising: (a) crossing at least one of inbredmaize parent plants GE024207702 and GE2543998, representative seed ofwhich have been deposited under ATCC Accession Nos: as PTA-XXXX andPTA-XXXX respectively, with plants of another maize line that comprise adesired trait to produce F1 progeny plants, wherein the desired trait isselected from the group consisting of male sterility, site-specificrecombination, increased transformability and abiotic stress tolerance;(b) selecting said F1 progeny plants that have the desired trait toproduce selected F1 progeny plants; (c) backcrossing the selectedprogeny plants with said inbred maize parent plant to produce backcrossprogeny plants; (d) selecting for backcross progeny plants that have thedesired trait and morphological and physiological characteristics ofsaid inbred maize parent plant to produce selected backcross progenyplants; (e) repeating steps (c) and (d) three or more times insuccession to produce a selected fourth or higher backcross progenyplants; and (f) crossing said fourth or higher backcross progeny plantwith the other inbred maize parent plant to produce a hybrid maizevariety 32H20 that comprises the desired trait and all of themorphological and physiological characteristics of hybrid maize variety32H20 listed in Table 1 as determined at the 5% significance level whengrown in the same environmental conditions.
 15. A plant produced by themethod of claim 14, wherein the plant has the desired trait and all ofthe physiological and morphological characteristics of hybrid maizevariety 32H20 listed in Table 1 as determined at the 5% significancelevel when grown in the same environmental conditions.
 16. The plant ofclaim 15, wherein the desired trait is male sterility and the trait isconferred by a nucleic acid molecule that confers male sterility. 17.The plant of claim 15, wherein the desired trait is site-specificrecombination and the site-specific recombination is conferred by amember of the group consisting of flp/frt, cre/lox, Gin, Pin, and R/RS.18. The plant of claim 15, wherein the desired trait is increasedtransformability and the trait is conferred by inbred maize line Hi-II.19. The plant of claim 15, wherein the desired trait is abiotic stresstolerance and the trait is conferred by a member of the group consistingof cytokinin, ethylene, abscisic acid, CBF, and a transcription factor.20. A process of introducing a desired trait into a hybrid maize variety32H20 comprising: (a) crossing at least one of inbred maize parentplants GE024207702 and GE2543998, representative seed of which have beendeposited under ATCC Accession Nos: as PTA-XXXX and PTA-XXXXrespectively, with plants of another maize line that comprise a desiredtrait to produce F1 progeny plants, wherein the desired trait isselected from the group consisting of herbicide resistance, insectresistance, and disease resistance; (b) selecting said F1 progeny plantsthat have the desired trait to produce selected F1 progeny plants; (c)backcrossing the selected progeny plants with said inbred maize parentplant to produce backcross progeny plants; (d) selecting for backcrossprogeny plants that have the desired trait and morphological andphysiological characteristics of said inbred maize parent plant toproduce selected backcross progeny plants; (e) repeating steps (c) and(d) three or more times in succession to produce a selected fourth orhigher backcross progeny plants; and (f) crossing said fourth or higherbackcross progeny plant with the other inbred maize parent plant toproduce a hybrid maize variety 32H20 that comprises the desired traitand all of the morphological and physiological characteristics of hybridmaize variety 32H20 listed in Table 1 as determined at the 5%significance level when grown in the same environmental conditions. 21.A plant produced by the method of claim 20, wherein the plant has thedesired trait and all of the physiological and morphologicalcharacteristics of hybrid maize variety 32H20 listed in Table 1 asdetermined at the 5% significance level when grown in the sameenvironmental conditions.
 22. The plant of claim 21, wherein the desiredtrait is herbicide resistance and said herbicide is glyphosate,glufosinate, a sulfonylurea herbicide, an imidazolinone herbicide, ahydroxyphenylpyruvate dioxygenase inhibitor or a protoporphyrinogenoxidase inhibitor.
 23. The plant of claim 21, wherein the desired traitis insect resistance and said insect resistance is conferred by anucleic acid molecule encoding a Bacillus thuringiensis endotoxin. 24.The plant of claim 21, wherein the desired trait is disease resistanceand the disease is caused by a bacterium, fungus, nematode or virus. 25.The plant of claim 21, wherein the desired trait is disease resistanceand the disease is caused by Colletotrichum graminiola.
 26. A process ofintroducing altered grain characteristics into a hybrid maize variety32H20 comprising: (a) crossing at least one of inbred maize parentplants GE024207702 and GE2543998, representative seed of which have beendeposited under ATCC Accession Nos: as PTA-XXXX and PTA-XXXXrespectively, with plants of another maize line that comprise a desiredtrait to produce F1 progeny plants, wherein the desired trait isselected from the group consisting of altered phosphorus, antioxidants,fatty acids, essential amino acids and carbohydrates; (b) selecting saidF1 progeny plants that have said nucleic acid molecule to produceselected F1 progeny plants; (c) backcrossing the selected progeny plantswith said inbred maize parent plant to produce backcross progeny plants;(d) selecting for backcross progeny plants that have said nucleic acidmolecule and morphological and physiological characteristics of saidinbred maize parent plant to produce selected backcross progeny plants;(e) repeating steps (c) and (d) three or more times in succession toproduce a selected fourth or higher backcross progeny plants; and (f)crossing said fourth or higher backcross progeny plant with the otherinbred maize parent plant to produce a hybrid maize variety 32H20 thatcomprises the desired trait and all of the morphological andphysiological characteristics of hybrid maize variety 32H20 listed inTable 1 as determined at the 5% significance level when grown in thesame environmental conditions.
 27. A plant produced by the method ofclaim 26, wherein the plant has the desired trait and all of thephysiological and morphological characteristics of hybrid maize variety32H20 listed in Table 1 as determined at the 5% significance level whengrown in the same environmental conditions.
 28. The plant of claim 27,wherein the desired trait is altered carbohydrate and the carbohydrateis waxy starch.
 29. The plant of claim 27, wherein said altered graincharacteristics are conferred by a nucleic acid molecule selected fromthe group consisting of NTR, TRX, dek1, TUSC 27, cs27, en27, Ipa1, Ipa3,mi1ps, hpt and hggt.
 30. A plant according to claim 2, wherein saidplant is modified by the addition of one mutant or transgenic gene thatconfers a characteristic selected from the group consisting of herbicideresistance, insect resistance or disease resistance.
 31. A plantaccording to claim 2, wherein said plant is modified by the addition ofone mutant or transgenic gene that confers a characteristic selectedfrom the group consisting of altered phosphorus, antioxidants, fattyacids, essential amino acids and carbohydrates.
 32. A plant according toclaim 2, wherein said plant is modified by the addition of one mutant ortransgenic gene that confers a characteristic selected from the groupconsisting of male sterility, site-specific recombination, increasedtransformability and abiotic stress tolerance.